Generation of glucose-responsive beta cells

ABSTRACT

The present invention relates to a method for generating glucose-responsive beta cells.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/852,900, filed Dec. 22, 2017, which claims priority under 35 U.S.C. §119 or 365 to Canadian Patent Application No. 2,983,845, filed Oct. 26,2017. The entire content of each application is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a method for generatingglucose-responsive beta cells from human pluripotent stem cells andhuman pancreatic progenitor cells.

BACKGROUND OF THE INVENTION

Cell therapy treatment of insulin dependent diabetes is facilitated bythe production of unlimited numbers of pancreatic cells that can andwill be able to function similarly to human islets. Accordingly, thereis a need for producing these pancreatic cell types derived from humanembryonic stem (hES) cells, as well as reliable methods for purifyingsuch cells. For example, the use of insulin-producing β-cells derivedfrom human embryonic stem cells (hESCs) would offer a vast improvementover current cell therapy procedures that utilize cells from donorpancreases. Currently cell therapy treatments for diabetes mellitus,such as type 1 or type 2 diabetes, which utilize cells from donorpancreases, are limited by the scarcity of high quality islet cellsneeded for transplant. For example, cell therapy for a single type 1diabetic patient requires a transplant of approximately 8×10⁸ pancreaticislet cells (Shapiro et al, 2000, N Engl J Med 343:230-238; Shapiro etal, 2001a, Best Pract Res Clin Endocrinol Metab 15:241-264; Shapiro etal, 2001b, British Medical Journal 322:861). As such, at least twohealthy donor organs are required to obtain sufficient islet cells for asuccessful transplant.

Embryonic stem (ES) cells thus represent a powerful model system for theinvestigation of mechanisms underlying pluripotent stem cell biology anddifferentiation within the early embryo, as well as providingopportunities for genetic manipulation of mammals and resultantcommercial, medical and agricultural applications. Furthermore,appropriate proliferation and differentiation of ES cells canpotentially be used to generate an unlimited source of cells suited totransplantation for treatment of diseases that result from cell damageor dysfunction. Other pluripotent stem cells and cell lines includingearly primitive ectoderm-like (EPL) cells, in vivo or in vitro derivedICM/epiblast, in vivo or in vitro derived primitive ectoderm, primordialgerm cells (EG cells), teratocarcinoma cells (EC cells), and pluripotentstem cells derived by dedifferentiation or by nuclear transfer can alsobe used.

Accordingly, there is a need for methods for generatingglucose-responsive beta cells.

SUMMARY OF THE INVENTION

The present invention provides methods for generating glucose-responsivebeta cells from pluripotent stem cells and human pancreatic progenitorcells.

In one aspect is provided a method of generating beta cells, comprisingthe steps of providing a starting cell population comprising at leastone cell capable of differentiation; wherein the cell capable ofdifferentiation is a pluripotent stem cell or a pancreatic progenitorcell expressing PDX1 and NKX6.1, wherein:

-   -   a. If the cell capable of differentiation is a pluripotent stem        cell, the method comprises the steps of:    -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration, thereby differentiating at least part of the cell        population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration, thereby further        differentiating the cell population into definitive endoderm        cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration,        thereby differentiating at least part of the cell population        into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration, thereby differentiating at least part of        the cell population into posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration, thereby differentiating        at least part of the cell population into early pancreatic        progenitor cells; and    -   vi) Incubating the cell population of v) in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        and human Noggin for a duration, thereby differentiating at        least part of the cell population into mature pancreatic        progenitor cells; and    -   vii) Further incubating the cell population of vi) for an        additional duration, thereby differentiating at least part of        the cell population into beta cells;    -   or    -   b. If the cell capable of differentiation is a pancreatic        progenitor cell, the method comprises the steps of:    -   viii) Incubating the starting cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration; and    -   ix) Incubating the cell population obtained in step viii) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration.

In another aspect is provided a population of cells obtainable by themethods disclosed herein, for treatment of a metabolic disorder in anindividual in need thereof.

In another aspect is provided a method of treatment of a metabolicdisorder in an individual in need thereof, wherein the method comprisesa step of providing a population of beta cells obtainable any of themethods disclosed herein and transplanting said population of beta cellsinto said individual.

The methods for obtaining glucose-responsive beta cells described hereinpresent several advantages: they are faster and use fewer factors (suchas differentiation factors) and are thus more reproducible than theprotocols known in the art. They are versatile and can be used ondifferent cell lines. The present methods are thus well suited forautomation, and are thereby envisioned to significantly reducemanufacturing costs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G. Analysis of in vitro differentiated PDXeG hESCs. FIG. 1A)Two differentiation protocols were used to obtain either pancreaticendoderm cells co-expressing PDX1 and NKX6.1 (protocol A (PEC)) orposterior foregut cells expressing PDX1 but lacking NKX6.1 (protocol B(PFG)). FIG. 1B) Schematic depicting the differentiation protocolreferred to as “protocol A”, generating PECs. FIG. 1C) FACS isolation ofGFP⁺ and GFP⁻ fractions at day 17 in hESCs treated according to protocolA. FIG. 1D) Gene expression analysis of sorted GFP⁺ and GFP⁻ cellsshowed significant enrichment of PE markers (importantly PDX1 andNKX6.1) in the GFP⁺ cells. The graphs depict mean expression±SEM (n=5)and represent the fold increase compared to control samples (GFP⁻ cells)at day 17. The control sample was arbitrarily set to a value of one.**p≤0.01, ***p≤0.001, and ****p≤0.0001. FIG. 1E) Schematic depicting thedifferentiation protocol referred to as “protocol B”, generating PFGcells. FIG. 1F) FACS isolation of GFP⁺ and GFP⁻ cells (from day 17)obtained by protocol B. FIG. 1G) Gene expression analysis of sorted GFP⁺and GFP⁻ cells showed that whereas markers such as PDX1, CDH1, ONECUT1,and SOX9 were enriched in the GFP⁺ cells, neither NKX6.1 nor MNX1 weresignificantly up-regulated in the GFP⁺ cells. The graphs depict meanexpression±SEM (n=2-4) and represent the fold increase compared tocontrol samples (GFP⁻ cells) at day 17. * p≤0.05, ** p≤0.01. See alsoFigure S1 of Ameri et al. (2017).

FIGS. 2A-2E. Global gene expression analysis of in vitro derivedPDX1+/NKX6.1+ PECs vs. PDX1⁺/NKX6.1⁻ cells. FIG. 2A) Heat-map displayinghierarchical clustering of genes differentially expressed in thePDX1⁺/NKX6.1⁺ (PEC, GFP+) pancreatic progenitors generated usingprotocol A, PDX1⁺/NKX6.1⁻ (PFG, GFP⁺) posterior foregut cells generatedusing protocol B, and PDX1⁻ (GFP⁻) cells from protocol A. FIG. 2B) Venndiagrams showing the distribution of genes up-regulated in PECs versusGFP⁻ cells, PECs versus PFG cells, and PFG cells versus GFP⁻ cells atday 17. FIG. 2C) Gene ontology (GO) analysis showing enrichment of genesin the PDX1⁺/NKX6.1⁺ pancreatic endoderm cells. Representative GOcategories are shown and plotted against −log (p-value). FIG. 2D)Expression of common genes expressed in the PFG and PE was analyzed inthe different sub-populations. FIG. 2E) Hierarchical clustering of thegenes differentially expressed in the 3-comparison analysis depicted inA (average expression levels are shown). The bars indicate sub-clusterswith relevant genes; nine different sub-clusters were created in total.Sub-cluster 3a shows genes enriched in the GFP⁻ cell population,including the novel cell surface marker CD49d (ITGA4), whereassub-cluster 5 displays genes enriched in the pancreatic endoderm cells(PEC cell fraction), also including the novel cell surface marker GP2.Sub-cluster 6 indicates genes enriched in PDX1⁺ cells irrespective ofNKX6.1 expression (PFG and PEC cells), such as CDH1 (ECAD), EPCAM, F3(CD142) and the novel cell surface marker FOLR1. See also Table S1 ofAmeri et al. (2017).

FIGS. 3A-3G. Validation of the novel cell surface markers GP2 and ITGA4in hESCs and human fetal pancreas. FIG. 3A) Flow cytometry analysis ofthe cell surface markers GP2, and ITGA4 performed on differentiatedhESCs cultured on MEFs (day 17), confirmed that GP2 was highly expressedin the GFP⁺ cells whereas ITGA4 was enriched in the GFP⁻ cells. FIG. 3B)Gene expression analysis showed that PE markers were highly enriched inGP2⁺/ITGA4⁻ sorted cells. The data are shown as mean expression±SEM(n=3), * p≤0.05, ** p≤0.01, ***p≤0.001. FIG. 3C) Flow cytometry analysisof GP2 and ITGA4 in genetically untagged HUES4 cells, cultured in afeeder-free system using protocol A depicted in FIG. 1. FIG. 3D)GP2⁺ITGA4⁻, ITGA4⁺GP2⁻, and GP2⁻ITGA4⁻ cells were sorted and the geneexpression pattern was analyzed. PDX1, SOX9, MNX1, and NKX6.1 weresignificantly enriched in the GP2⁺ITGA4⁻ cell fractions. The remainingPDX1⁺ cells in the GP2⁻ITGA4⁻ fraction express only low levels ofNKX6.1, confirming that GP2 specifically enrich for PDX1⁺/NKX6.1⁺ cells.The data are shown as mean expression±SEM (n=5-6), * p≤0.05, ** p≤0.01,*** p≤0.001, and **** p≤0.0001. FIG. 3E) Flow cytometry analysis of GP2and ITGA4 expression in human fetal pancreas (9.1 WD) gated onnon-hematopoietic and non-endothelial cells (CD45⁻CD31⁻). FIG. 3F) qPCRanalysis of PDX1, and NKX6.1 expression in FACS sorted GP2⁺ and ITGA4⁺cell populations, showed significant enrichment of PDX1 and NKX6.1 inthe GP2⁺ vs. the ITGA4⁺ cells. Results are shown as mean expression±SD,presented in arbitrary units (AU) relative to expression of the controlgene PPIA. *p=0.023 and **p=0.010. ND=Non-Detected. FIG. 3G) Flowcytometry analysis of PDX1 and NKX6.1 expression in GP2⁺ and CD45⁺/CD31⁺cells at 8.7WD. 91% of the GP2⁺/PDX1⁺ cells co-expressed NKX6.1.CD45⁺CD31⁺ cells were used as a negative control for PDX1 and NKX6.1expression. FACS plots are representative of 3 independent experiments.See also Figure S2 of Ameri et al. 2017.

FIGS. 4A-4G. Validation of GP2 using an independent and previouslypublished differentiation protocol. FIG. 4A) Scheme for generation ofhPSC-derived PECs according to a modified protocol by Rezania et. al,2013. AA: Activin A, F7: FGF7, Nog: Noggin, DF12: DMEMF12, VitC: vitaminC. FIG. 4B) Characterization of GFP and GP2 expression by flow cytometryon differentiated PDXeG cells. FIG. 4C) qPCR analysis of the sortedpopulations: GP2⁻GFP⁻, GP2⁻GFP⁺, and GP2⁺GFP⁺ cells showed that NKX6.1expression is significantly enriched in the GP2⁺GFP⁺ cell fraction incomparison to the GP2⁻GFP⁺ cell fraction. The data are shown as meanexpression±SEM (n=3). FIG. 4D) Immunofluorescence stainings of thesorted cell populations confirmed significant enrichment ofPDX1⁺/NKX6.1⁺ cells in the GP2⁺/GFP⁺ cells. Scale bars, 100 μm. FIG. 4E)Flow cytometry analysis of differentiated PDXeG cells on day 13. FIG.4F) PDX1 and NKX6.1 expression in cultures at day 13 was analyzed byimmunofluorescence. Scale bars, 100 μm. FIG. 4G) Percentage ofPDX1⁺/NKX6.1⁺ quantified from day 13 cultures. See also Figure S3 ofAmeri et al. 2017.

FIGS. 5A-5H. Differentiation of purified GP2⁺/ITGA4⁻ PECs into glucoseresponsive insulin expressing cells. FIG. 5A) Schematic illustratingdifferentiation of hESCs into PECs that are dissociated and stained withthe cell surface markers ITGA4 and GP2. FIG. 5B) A table depicting thedifferentiation protocol to generate insulin expressing cells from PECs.Rocki is omitted when the protocol is applied to unsorted cultures.Rocki: Rock inhibitor, For: Forskolin, Alki: Alk5 inhibitor, Nog:Noggin, Nic: Nicotinamide, DF12: DMEM/F-12, B27: B27 Supplement. FIG.5C) Flow cytometry analysis of differentiated PECs (from day 18) stainedwith GP2 and ITGA4. FIG. 5D) C-peptide staining of re-plated GP2^(High)and GP2^(Low) expressing cells. Scale bars, 100 μm. FIG. 5E) Percentageof CPEP⁺ cells in the GP2^(High) and GP2^(Low) cells is shown. ****p≤0.0001. FIG. 5F) Immunofluorescence analysis of FACS sortedGP2⁺/ITGA4⁻ pancreatic endoderm cells re-plated and differentiated toinsulin expressing cells. Scale bars, 100 μm. FIG. 5G) Percentage ofGLU⁺ cells in the unsorted and GP2^(High) cells is shown. ****p≤0.0001.FIG. 5H) The release of human C-peptide was measured in thedifferentiated GP2⁺/ITGA4⁻ cells by a static glucose-stimulated insulinsecretion assay (GSIS). Error bars represent mean expression±SEM (n=4),***p≤0.001, and **** p≤0.0001. See also FIGS. 8A-8H.

FIGS. 6A-6H. Validation of GP2 in the GMP graded cell line MShef-7. FIG.6A) Time course analysis of INS and GLU expression in differentiatedMShef-7 cells. The data are shown as mean expression±SEM (n=3). FIG. 6B)Flow cytometry analysis of differentiated PECs stained with GP2 andITGA4. FIG. 6C) Co-staining of CPEP (white) and GLU (green) of unsortedcells. Scale bar, 100 μm FIGS. 6D-6E) Immunostainings of differentiatedGP2^(Low) cells (FIG. 6D) and GP2^(High) cells (FIG. 6E) with CPEP(white), GLU (green), and NKX6.1 (red). Scale bars, 100 μm. FIG. 6F)Percentage of CPEP⁺ cells in unsorted, GP2^(Low), GP2^(High) cells.****p≤0.0001 FIG. 6G) Percentage of GLU⁺ cells in unsorted, GP2^(Low)GP2^(High) cells. ****p≤0.0001. FIG. 6H) Static GSIS assay ofdifferentiated GP2^(High) cells showed a 2-fold change in CPEP response.Error bars represent mean expression±SEM ** p≤0.01. See also Figure S5of Ameri et al. (2017).

FIGS. 7A-7M. CDKN1A and CDKN2A knockdown promote proliferation ofhESC-derived PECs. FIGS. 7A-7B) Cell cycle analysis of differentiatedhESCs at day 14 corresponding to early PECs. Cells from day 11 weretransfected with CDKN1A siRNA and harvested 72 h later, stained with EDUand analyzed by flow cytometry (a representative analysis is shown).FIG. 7C) Summary of data depicted in FIGS. 7A-7B, where thecorresponding ratio of CDKN1A/CTR siRNA for each cell cycle phase isshown. FIG. 7D) qPCR analysis of samples treated with scrambled andCDKN1A siRNA confirmed up-regulation of MKI67 expression 24 h afterCDKN1A knockdown. The data are shown as mean expression±SEM,****p<0.0001. FIG. 7E) Immunofluorescence analysis confirmed asignificant increase of MKI67⁺ cells 72 h after knockdown of CDKN1A.Scale bars, 100 μm. FIG. 7F) Quantification of MKI67 expressing cells inthe cultures showed there was a significant increase in the number ofMKI67⁺ cells, ****p<0.0001. FIGS. 7G-7H) Cells from day 11 weretransfected with CDKN2A siRNA and harvested 72 h later, stained with EDUand analyzed by flow cytometry (a representative analysis is shown).FIG. 71) Summary of data depicted in FIGS. 7K-7L, where thecorresponding ratio of CDKN2A/CTR siRNA for each cell cycle phase isshown. FIG. 7J) qPCR analysis showed no statistically significantup-regulation of MKI67 expression in the CDKN2A knocked down samples 24h after transfection, error bars represent mean expression±SEM. However,immunofluorescence analysis showed a significant increase of MKI67⁺cells (FIG. 7K) after 72 h of knockdown of CDKN2A. Scale bars, 100 μm.FIG. 7L) Quantification of MKI67 expressing cells in the culturesconfirmed the significant increase in the number of MKI67⁺ cells,****p<0.0001. FIG. 7M) Schematic displaying PE formation duringdevelopment. As the PE cells mature, CDKN1A (p21) and CDKN2A (p16)expression levels increase and MK167 expression is down-regulated (upperpanel). Downregulation of p21 or p16 within early PECs prevents thedecrease in proliferation during PE maturation (middle panel), whereasinhibition within late PE is unable to restore proliferation (lowerpanel). See also Figure S6 and S7 of Ameri et al. (2017).

FIGS. 8A-8H. Differentiation of hESCs into glucose responsive insulinexpressing cells. FIG. 8A) Schematic illustrating the differentiationprotocol (referred to as protocol C) for generating hPSC-derived insulinproducing cells. AA: Activin A, CHIR: CHIR99021, RA: Retinoic acid, F2:FGF2, TPB:((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam),Nog: Noggin, For: Forskolin, Alki: Alk5 inhibitor, Nic: Nicotinamide,RPMI: RPMI-1640 DF12: DMEM/F-12, B27: B27 Supplement. FIG. 8B)Immunofluorescence stainings of differentiated cells at the PE stage(day 18). Scale bar, 100 μm. FIG. 8C) Quantification of PDX1⁺/NKX6-1⁺cells at day 18. FIG. 8D) GP2 and ITGA4 staining of PECs from day 18.FIG. 8E) Time course analysis of INS and GLU expression indifferentiated HUES4 cells. The data are shown as mean expression±SEM(n=3). FIG. 8F) Immunofluorescence stainings of late stage cultures ofdifferentiated hESCs. Scale bars, 100 μm. FIG. 8G) High magnificationimages of the immunofluorescence staining from FIG. 8F). Scale bars, 50μm FIG. 8H) Released human C-peptide levels were measured by a staticGSIS assay in differentiated hESCs at d32. Error bars represent ±SEM.n=3), **p<0.01, ****p<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified a method of generating beta cells.

The most recent success in generating hPSC-derived glucose responsiveinsulin-producing cells that share functional properties with normalbeta cells (Pagliuca et al., 2014, Rezania et al., 2014), have made theimplementation of a cell-based therapy for the treatment of type Idiabetes a palpable reality. The therapeutic success of this approachwill depend on the ability to upscale production of hPSC-derivatives.Estimates put the number of functional cells required for organ repairand disease recovery in the order of 10⁹ per patient (Pagliuca et al.,2014, Kempf et al., 2016). Thus, differentiation strategies will need tobe adapted for mass production at an industrial scale. Currently,generation of glucose-responsive insulin-producing cells requirestedious and complicated multistep protocols, where undifferentiatedhPSCs with tumorigenic propensity are used as the starting cellpopulation. By establishing strategies where more mature cells are usedfor generating beta cells, the potential contamination withtumor-causing cells in the final cell preparation to be used for celltherapy could be prevented, and safer and more reproduciblemanufacturing procedures could be achieved. However, stage-specificsurface markers that can be used to purify late stage cell populationsduring pancreatic differentiation are lacking.

During normal embryonic development the highly proliferative human andmouse pancreatic progenitors, recognized by their co-expression of thetranscription factors Pancreatic duodenal homeobox 1 (PDX1) and NK6homeobox 1 (NKX6.1), are responsible for the proper growth of thepancreatic epithelium and give rise to all the pancreatic cell typesincluding exocrine, ductal, and endocrine cells. Consequently,pancreatic progenitor cells could serve as an ideal starting populationfor the generation of hormone producing endocrine cells such as the betacells. Furthermore, previous publications support the notion thatenrichment of pancreatic progenitors would reduce the risk of teratomaformation upon transplantation. Isolation of hPPCs could be obtainedusing tissue-specific cell surface molecules, and in fact markers forhPSC-derived pancreatic cell populations (CD142 for pancreaticprogenitors and CD200/CD318 for endocrine cells) have been reported(Kelly 2011). However, the specificity of the pancreatic progenitormarker CD142 was questionable, as the populations enriched with thismolecule were not exclusively composed of pancreatic progenitor cells aspointed out by the authors. Hence, the need for new and more specificmarkers to enrich for a progenitor population remains to be fulfilled.

Generation of tissue specific reporter cell lines could aid in theprocess of identifying pancreas-specific cell surface markers. Thus weestablished a PDX1-eGFP reporter cell line (PDXeG) by gene targeting inorder to enable the isolation of pure PDX1⁺ pancreatic progenitor cellsfrom hPSCs. By using the PDXeG reporter cell line as a genetic tool, wewere able to isolate different subpopulations of PDX1⁺ cells and performa genome wide expression analysis that allowed us to identify novel cellsurface markers for isolation of hPPCs. Specifically, we identifiedthree novel cell surface markers allowing us to separate true pancreaticprogenitors from posterior foregut endoderm cells: glycoprotein 2(zymogen granule membrane) (GP2) as a marker for isolation ofPDX1⁺/NKX6.1⁺ hPPCs, Integrin Alpha-4 (ITGA4 or CD49d) as a negativeselection marker labeling the PDX1− cell fraction, and finally a thirdmarker, Folate receptor 1 (adult) (FOLR1) recognizing the PDX1⁺/NKX6.1−cells.

The specificity of these markers was demonstrated by using human fetalpancreas tissue, as described in patent application WO 2016/170069.

In one aspect is provided a method of generating beta cells, comprisingthe steps of providing a starting cell population comprising at leastone cell capable of differentiation; wherein the cell capable ofdifferentiation is a pluripotent stem cell or a pancreatic progenitorcell expressing PDX1 and NKX6.1, wherein:

-   -   a. If the cell capable of differentiation is a pluripotent stem        cell, the method comprises the steps of:    -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration, thereby differentiating at least part of the cell        population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration, thereby further        differentiating the cell population into definitive endoderm        cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration,        thereby differentiating at least part of the cell population        into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration, thereby differentiating at least part of        the cell population into posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration, thereby differentiating        at least part of the cell population into early pancreatic        progenitor cells; and    -   vi) Incubating the cell population of v) in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        and human Noggin for a duration, thereby differentiating at        least part of the cell population into mature pancreatic        progenitor cells; and    -   vii) Further incubating the cell population of vi) for an        additional duration, thereby differentiating at least part of        the cell population into beta cells; or    -   b. If the cell capable of differentiation is a pancreatic        progenitor cell, the method comprises the steps of:    -   viii) Incubating the starting cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration; and    -   ix) Incubating the cell population obtained in step viii) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration.

Also disclosed is a method of generating beta cells, comprising thesteps of providing a starting cell population comprising at least onecell capable of differentiation; wherein the cell capable ofdifferentiation is a pluripotent stem cell or a pancreatic progenitorcell expressing PDX1 and NKX6.1, wherein:

-   -   a. If the cell capable of differentiation is a pluripotent stem        cell, the method comprises the steps of:    -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration of one day, thereby differentiating at least part of        the cell population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration of between 3 and 6 days,        thereby further differentiating the cell population into        definitive endoderm cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration of        between 3 and 6 days, thereby differentiating at least part of        the cell population into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration of between 3 and 6 days, thereby        differentiating at least part of the cell population into        posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration of between 3 and 6 days,        thereby differentiating at least part of the cell population        into early pancreatic progenitor cells; and    -   vi) Incubating the cell population of v) in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        and human Noggin for a duration of between 3 and 6 days, thereby        differentiating at least part of the cell population into mature        pancreatic progenitor cells; and    -   vii) Further incubating the cell population of vi) for an        additional 7 to 23 days, thereby differentiating at least part        of the cell population into beta cells; or    -   b. If the cell capable of differentiation is a pancreatic        progenitor cell, the method comprises the steps of:    -   viii) Incubating the starting cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration of between 1 and        2 days; and

Incubating the cell population obtained in step viii) in DMEM/F12 mediumcomprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide, humanNoggin without Rock inhibitor for a duration of between 7 and 14 days.

Accordingly, a method of generating beta cells is disclosed, said methodcomprising the steps of providing a starting cell population comprisingat least one cell capable of differentiation, wherein the cell capableof differentiation is a pluripotent stem cell, and further comprisingthe steps of:

-   -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration of one day, thereby differentiating at least part of        the cell population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration of between 3 and 6 days,        thereby further differentiating the cell population into        definitive endoderm cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration of        between 3 and 6 days, thereby differentiating at least part of        the cell population into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration of between 3 and 6 days, thereby        differentiating at least part of the cell population into        posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration of between 3 and 6 days,        thereby differentiating at least part of the cell population        into early pancreatic progenitor cells; and    -   vi) Incubating the cell population of v) in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        and human Noggin for a duration of between 3 and 6 days, thereby        differentiating at least part of the cell population into mature        pancreatic progenitor cells; and    -   vii) Further incubating the cell population of vi) for an        additional 7 to 23 days, thereby differentiating at least part        of the cell population into beta cells.

Also disclosed is a method of generating beta cells, comprising thesteps of providing a starting cell population comprising a pancreaticprogenitor cell expressing PDX1 and NKX6.1, wherein the method furthercomprises the steps of:

-   -   viii) Incubating the starting cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration of between 1 and        2 days; and    -   ix) Incubating the cell population obtained in step viii) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration of between 7 and 14 days.

In some embodiments, the method of generating beta cells comprises thesteps of providing a starting cell population comprising at least onecell capable of differentiation, wherein the cell capable ofdifferentiation is a pluripotent stem cell, and further comprising thesteps of:

-   -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration, thereby differentiating at least part of the cell        population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration, thereby further        differentiating the cell population into definitive endoderm        cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration,        thereby differentiating at least part of the cell population        into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration, thereby differentiating at least part of        the cell population into posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration, thereby differentiating        at least part of the cell population into early pancreatic        progenitor cells, and enriching the obtained cell population for        cells expressing PDX1 and NKX6.1 as described herein, thereby        obtaining an enriched cell population;    -   vi) Incubating the enriched cell population of step v) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin and Rock inhibitor for a        duration; and    -   vii) Incubating the cell population obtained in step vi) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration.

In some embodiments, the method of generating beta cells comprises thesteps of providing a starting cell population comprising at least onecell capable of differentiation, wherein the cell capable ofdifferentiation is a pluripotent stem cell, and further comprising thesteps of:

-   -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration of one day, thereby differentiating at least part of        the cell population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration of between 3 and 6 days,        thereby further differentiating the cell population into        definitive endoderm cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration of        between 3 and 6 days, thereby differentiating at least part of        the cell population into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration of between 3 and 6 days, thereby        differentiating at least part of the cell population into        posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration of between 3 and 6 days,        thereby differentiating at least part of the cell population        into early pancreatic progenitor cells, and enriching the        obtained cell population for cells expressing PDX1 and NKX6.1 as        described herein, thereby obtaining an enriched cell population;    -   vi) Incubating the enriched cell population of step v) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin and Rock inhibitor for a        duration of between 1 and 2 days; and    -   vii) Incubating the cell population obtained in step vi) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration of between 7 and 14 days.

In other embodiments, the method of generating beta cells comprises thesteps of providing a starting cell population comprising at least onecell capable of differentiation, wherein the cell capable ofdifferentiation is a pluripotent stem cell, and further comprising thesteps of:

-   -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration, thereby differentiating at least part of the cell        population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration, thereby further        differentiating the cell population into definitive endoderm        cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration,        thereby differentiating at least part of the cell population        into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration, thereby differentiating at least part of        the cell population into posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration, thereby differentiating        at least part of the cell population into early pancreatic        progenitor cells, and further incubating the early pancreatic        progenitor cell population in DMEM/F12 medium comprising        B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide, and human        Noggin for a duration, thereby differentiating at least part of        the cell population into mature pancreatic progenitor cells, and        enriching the obtained cell population for cells expressing PDX1        and NKX6.1 as described herein, thereby obtaining an enriched        cell population;    -   vi) Incubating the enriched cell population of step v) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin and Rock inhibitor for a        duration; and    -   vii) Incubating the cell population obtained in step vi) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration.

In other embodiments, the method of generating beta cells comprises thesteps of providing a starting cell population comprising at least onecell capable of differentiation, wherein the cell capable ofdifferentiation is a pluripotent stem cell, and further comprising thesteps of:

-   -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration of one day, thereby differentiating at least part of        the cell population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration of between 3 and 6 days,        thereby further differentiating the cell population into        definitive endoderm cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration of        between 3 and 6 days, thereby differentiating at least part of        the cell population into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration of between 3 and 6 days, thereby        differentiating at least part of the cell population into        posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration of between 3 and 6 days,        thereby differentiating at least part of the cell population        into early pancreatic progenitor cells, and further incubating        the early pancreatic progenitor cell population in DMEM/F12        medium comprising B27+insulin, Forskolin, Alk5 inhibitor,        Nicotinamide, and human Noggin for a duration of between 3 and 6        days, thereby differentiating at least part of the cell        population into mature pancreatic progenitor cells, and        enriching the obtained cell population for cells expressing PDX1        and NKX6.1 as described herein, thereby obtaining an enriched        cell population;    -   vi) Incubating the enriched cell population of step v) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin and Rock inhibitor for a        duration of between 1 and 2 days; and    -   vii) Incubating the cell population obtained in step vi) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration of between 7 and 14 days.

In another aspect is provided a population of cells obtainable by themethods disclosed herein, for treatment of a metabolic disorder in anindividual in need thereof.

In another aspect is provided a method of treatment of a metabolicdisorder in an individual in need thereof, wherein the method comprisesa step of providing a population of beta cells obtainable any of themethods disclosed herein and transplanting said population of beta cellsinto said individual.

Definitions

Antibody. The term ‘antibody’ describes a functional component of serumand is often referred to either as a collection of molecules (antibodiesor immunoglobulin) or as one molecule (the antibody molecule orimmunoglobulin molecule). An antibody molecule is capable of binding toor reacting with a specific antigenic determinant (the antigen or theantigenic epitope), which in turn may lead to induction of immunologicaleffector mechanisms. An individual antibody molecule is usually regardedas monospecific, and a composition of antibody molecules may bemonoclonal (i.e., consisting of identical antibody molecules) orpolyclonal (i.e., consisting of different antibody molecules reactingwith the same or different epitopes on the same antigen or on distinct,different antigens). Each antibody molecule has a unique structure thatenables it to bind specifically to its corresponding antigen, and allnatural antibody molecules have the same overall basic structure of twoidentical light chains and two identical heavy chains. Antibodies arealso known collectively as immunoglobulins. The terms antibody orantibodies as used herein is used in the broadest sense and coversintact antibodies, chimeric, humanized, fully human and single chainantibodies, as well as binding fragments of antibodies, such as Fab,F(ab′)₂, Fv fragments or scFv fragments, as well as multimeric formssuch as dimeric IgA molecules or pentavalent IgM.

Antigen. An antigen is a molecule comprising at least one epitope. Theantigen may for example be a polypeptide, polysaccharide, protein,lipoprotein or glycoprotein.

Definitive endoderm. As used herein, “definitive endoderm” or “DE”refers to a multipotent cell that can differentiate into cells of thegut tube or organs derived from the gut tube. In accordance with certainembodiments, the definitive endoderm cells and cells derived therefromare mammalian cells, and in a preferred embodiment, the definitiveendoderm cells are human cells. In some embodiments, definitive endodermcells express or fail to significantly express certain markers. In someembodiments, one or more markers selected from SOX17, CXCR4, MIXLI, GATA4, FOXA2, GSC, FGF 17, VWF, CALCR, FOXQI, CMKORI, CER and CRIPI areexpressed in definitive endoderm cells. In other embodiments, one ormore markers selected from OCT4, HNF4A, alpha-fetoprotein (AFP),Thrombomodulin (TM), SPARC and SOX7 are not significantly expressed indefinitive endoderm cells. Definitive endoderm cells do not expressPDX-1.

Differentiable or differentiated cell. As used herein, the phrase,“differentiable cell” or “differentiated cell” or “hES-derived cell” canrefer to pluripotent, multipotent, oligopotent or even unipotent cells,as defined in detail below. In certain embodiments, the differentiablecells are pluripotent differentiable cells. In more specificembodiments, the pluripotent differentiable cells are selected from thegroup consisting of embryonic stem cells, ICM/epiblast cells, primitiveectoderm cells, primordial germ cells, and teratocarcinoma cells. In oneparticular embodiment, the differentiable cells are mammalian embryonicstem cells. In a more particular embodiment, the differentiable cellsare human embryonic stem cells. Certain embodiments also contemplatedifferentiable cells from any source within an animal, provided thecells are differentiable as defined herein. For example, differentiablecells can be harvested from embryos, or any primordial germ layertherein, from placental or chorion tissue, or from more mature tissuesuch as adult stem cells including, but not limited to adipose, bonemarrow, nervous tissue, mammary tissue, liver tissue, pancreas,epithelial, respiratory, gonadal and muscle tissue. In specificembodiments, the differentiable cells are embryonic stem cells. In otherspecific embodiments, the differentiable cells are adult stem cells. Instill other specific embodiments, the stem cells are placental- orchorionic-derived stem cells.

Differentiation. As used herein, the term “differentiation” refers tothe production of a cell type that is more differentiated than the celltype from which it is derived. The term therefore encompasses cell typesthat are partially and terminally differentiated. Similarly, “producedfrom hESCs,” “derived from hESCs,” “differentiated from hESCs,” “hESderived cell” and equivalent expressions refer to the production of adifferentiated cell type from hESCs in vitro and in vivo.

Embryonic. As used herein, “embryonic” refers to a range ofdevelopmental stages of an organism beginning with a single zygote andending with a multicellular structure that no longer comprisespluripotent or totipotent cells other than developed gametic cells. Inaddition to embryos derived by gamete fusion, the term “embryonic”refers to embryos derived by somatic cell nuclear transfer.

Expression level. As used herein, the term “expression level” can referto the level of transcript (mRNA) or to the level of protein for aparticular gene or protein, respectively. Expression levels can thus bedetermined by methods known in the art, by determining transcriptionlevel or protein level. Transcription levels can be measured byquantifying the amount of transcript by methods such as, but not limitedto, Northern blot, RT-PCR or microarray-based methods. Protein levelscan be measured by methods such as, but not limited to, Western blot andimmunostaining.

Human embryonic stem cells. The human embryonic stem cells are derivedfrom the undifferentiated inner cell mass of the human embryo. Thesecells are pluripotent and are able to differentiate into all derivativesof the three primary germ layers namely: ectoderm, endoderm and mesoderm(Thomson et al., 1998). As used herein, the term “human pluripotent stemcells” (hPS) refers to cells that may be derived from any source andthat are capable, under appropriate conditions, of producing humanprogeny of different cell types that are derivatives of all of the 3germinal layers (endoderm, mesoderm, and ectoderm). hPS cells may havethe ability to form a teratoma in 8-12 week old SLID mice and/or theability to form identifiable cells of all three germ layers in tissueculture. Included in the definition of human pluripotent stem cells areembryonic cells of various types including human blastocyst derived stem(hBS) cells in literature often denoted as human embryonic stem (hES)cells (see, e.g., Thomson et al. (1998), Heins et. al. (2004), as wellas induced pluripotent stem cells (see, e.g. Yu et al. (2007) Science318:5858; Takahashi et al. (2007) Cell 131 (5):861). The various methodsand other embodiments described herein may require or utilise hPS cells(hPSCs) from a variety of sources. For example, hPS cells suitable foruse may be obtained from developing embryos. Additionally oralternatively, suitable hPS cells may be obtained from established celllines and/or human induced pluripotent stem (hiPS) cells by methods,which do not require the destruction of embryos (Chung et al. 2008).

As used herein “hiPS cells” refers to human induced pluripotent stemcells. As used herein, the term “blastocyst-derived stem cell” isdenoted BS cell, and the human form is termed “hBS cells”. In literaturethe cells are often referred to as embryonic stem cells, and morespecifically human embryonic stem cells (hESCs). The pluripotent stemcells used in the present invention can thus be embryonic stem cellsprepared from blastocysts, as described in e.g. WO 03/055992 and WO2007/042225, or be commercially available hBS cells or cell lines.However, it is further envisaged that any human pluripotent stem cellcan be used in the present invention, including differentiated adultcells which are reprogrammed to pluripotent stem cells by e.g. thetreating adult cells with certain transcription factors, such as OCT4,SOX2, NANOG, and LIN28 as disclosed in Yu, et al., 2007, Takahashi etal. 2007 and Yu et al 2009.

Inactivation: The term ‘inactivation’ is herein used in connection withinactivation of the function of a given protein in a cell and refers tomanipulations of the cell in order to obtain a loss of function.Inactivation may be achieved as known in the art, e.g. by using aninhibitor capable of inhibiting the function of the protein.Inactivation can also be achieved by mutation or deletion of the geneencoding the protein. Silencing, for example by using siRNAs, can alsobe used to achieve inactivation, as known to the person skilled in theart. Inactivation may be transient or permanent. Inactivation may alsobe reversible or irreversible. For example, incubation of a cellpopulation with an inhibitor will typically result in transientinactivation for as long as the inhibitor is effective or present.Removing the inhibitor from the environment will generally result inalleviation of the inactivation. Likewise, siRNAs will typically onlyhave a silencing effect for as long as they are expressed or present.Deletion or mutation of a gene on the other hand will typically resultin permanent inactivation, although the person skilled in the art willknow how to reverse the effects of deletion or mutation, for example bygene editing methods.

Induced pluripotent stem cell. Induced pluripotent stem cells (or iPSCs)can be derived directly from adult cells by reprogramming (Takashashi etal., 2006). iPSCs can be induced by proteins and are then termedprotein-induced pluripotent stem cells (piPSCs).

Ligand. As used herein, “ligand” refers to a moiety or binding partnerthat specifically binds or cross-reacts to the marker or target orreceptor or membrane protein on the cell or to the soluble analyte in asample or solution. The target on the cell includes but is not limitedto a marker. Examples of such ligands include, but are not limited to,an antibody that binds a cellular antigen, an antibody that binds asoluble antigen, an antigen that binds an antibody already bound to thecellular or soluble antigen; a lectin that binds to a solublecarbohydrate or to a carbohydrate moiety which is a part of aglycoprotein or glycolipid; or functional fragments of such antibodiesand antigens that are capable of binding; a nucleic acid sequencesufficiently complementary to a target nucleic acid sequence of thecellular target or soluble analyte to bind the target or analytesequence, a nucleic acid sequence sufficiently complementary to a ligandnucleic acid sequence already bound to the cellular marker or target orsoluble analyte, or a chemical or proteinaceous compound, such as biotinor avidin. Ligands can be soluble or can be immobilized on the capturemedium (i.e., synthetically covalently linked to a bead), as indicatedby the assay format, e.g., antibody affinity chromatography. As definedherein, ligands include, but are not limited to, various agents thatdetect and react with one or more specific cellular markers or targetsor soluble analytes.

Marker. As used herein, “marker”, “epitope”, “target”, “receptor” orequivalents thereof can refer to any molecule that can be observed ordetected. For example, a marker can include, but is not limited to, anucleic acid, such as a transcript of a specific gene, a polypeptideproduct of a gene, such as a membrane protein, a non-gene productpolypeptide, a glycoprotein, a carbohydrate, a glycolipid, a lipid, alipoprotein or a small molecule (for example, molecules having amolecular weight of less than 10,000 amu). A “cell surface marker” is amarker present at the cell surface.

Multipotent cell. As used herein, “multipotent” or “multipotent cell”refers to a cell type that can give rise to a limited number of otherparticular cell types. Multipotent cells are committed to one or moreembryonic cell fates, and thus, in contrast to pluripotent stem cells,cannot give rise to each of the three germ layer lineages as well asextraembryonic cells.

Naïve stem cell and primed stem cell. Naïve stem cells have thepotential to develop into any kind of cell, unlike primed stem cells,which are able to differentiate into several types of cells but arealready predetermined to some extent. Naïve stem cells have been knownto exist in mice but human naïve stem cells have only been describedrecently (Takashima et al., 2014). Naïve stem cells can self-renewcontinuously without ERK signalling, are phenotypically stable, and arekaryotypically intact. They differentiate in vitro and form teratomas invivo. Metabolism is reprogrammed with activation of mitochondria)respiration as in ESC. The pluripotent state of human cells can be resetby short-term expression of two components, NANOG and KLF2, as describedin Takashima et al., 2014. Naive PSCs share many properties with theinner cell mass of the blastocyst, while the primed PSCs resembleepiblast cells of a more advanced, pregastrulating stage embryo. In themouse, the naive state is represented by embryonic stem cells (mESCs)and the primed state by epiblast stem cells (EpiSCs). In humans,blastocyst derived ESCs have been regarded until recently as the humanequivalent of mESCs. However, without being bound by theory, based onmultiple characteristics such as flat morphology, dependence on growthfactors, or X-chromosome inactivation, hESCs (and human inducedpluripotent stem cell (hiPSCs)) are closer to mouse EpiSCs than to mESCsand, as such, more likely correspond to the primed rather than the naivestate of pluripotency (Tesar et al. 2007; Stadtfeld and Hochedlinger2010).

Naturally occurring antibody. The term ‘naturally occurring antibody’refers to heterotetrameric glycoproteins capable of recognising andbinding an antigen and comprising two identical heavy (H) chains and twoidentical light (L) chains inter-connected by disulfide bonds. Eachheavy chain comprises a heavy chain variable region (abbreviated hereinas V_(H)) and a heavy chain constant region (abbreviated herein asC_(H)). Each light chain comprises a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region(abbreviated herein as CL). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FRs). Antibodies may compriseseveral identical heterotetramers.

Pancreatic progenitor cell (PPC) or multipotent pancreatic progenitorcell. A progenitor cell is a cell that is committed to differentiateinto a certain type of cell. Pancreatic progenitor cells are thusmultipotent and can differentiate and give rise to all cell types of thepancreas. The term ‘pancreatic progenitor cell’ or ‘true pancreaticprogenitor’ refers herein to a cell, which is capable of differentiatinginto all pancreatic lineages, including acinar, duct and endocrine, suchas insulin-producing cells. The term is herein used interchangeably withthe term pancreatic endoderm cells (PECs).

Partially mature cell. As used herein, “partially mature cells” refer tocells that exhibit at least one characteristic of the phenotype, such asmorphology or protein expression, of a mature cell from the same organor tissue. Some embodiments contemplate using differentiable cells fromany animal capable of generating differentiable cells, e.g., pancreatictype cells such as beta cells. The animals from which the differentiablecells are harvested can be vertebrate or invertebrate, mammalian ornon-mammalian, human or non-human. Examples of animal sources include,but are not limited to, primates, rodents, canines, felines, equines,bovines and porcines.

Pluripotent stem cell. By “pluripotent” is meant that the cell can giverise to each of the three germ layer lineages. Pluripotent stem cells,however, may not be capable of producing an entire organism. In certainembodiments, the pluripotent stem cells used as starting material arestem cells, including human embryonic stem cells. Pluripotent stem cellscan be derived by explanting cells from embryos at different stages ofdevelopment. PSCs (pluripotent stem cells) can be classified into twodistinct states, naive and primed, depending on which stage they areduring embryonic development.

Stem cell. A stem cell is an undifferentiated cell that candifferentiate into specialized cells and can divide to produce more stemcells. The term stem cell comprises embryonic stem cells, adult stemcells, naïve stem cells as well as induced pluripotent stem cells. Stemcells are defined by their ability at the single cell level to bothself-renew and differentiate to produce progeny cells, includingself-renewing progenitors, non-renewing progenitors, and terminallydifferentiated cells. Stem cells are also characterized by their abilityto differentiate in vitro into functional cells of various cell lineagesfrom multiple germ layers (endoderm, mesoderm and ectoderm), as well asto give rise to tissues of multiple germ layers followingtransplantation and to contribute substantially to most, if not all,tissues following injection into blastocysts. Stem cells are classifiedby their developmental potential as: (1) totipotent, meaning able togive rise to all embryonic and extraembryonic cell types; (2)pluripotent, meaning able to give rise to all embryonic cell types; (3)multipotent, meaning able to give rise to a subset of cell lineages, butall within a particular tissue, organ, or physiological system (forexample, hematopoietic stem cells (HSC) can produce progeny that includeHSC (self-renewal), blood cell restricted oligopotent progenitors andall cell types and elements (e.g., platelets) that are normal componentsof the blood); (4) oligopotent, meaning able to give rise to a morerestricted subset of cell lineages than multipotent stem cells; and (5)unipotent, meaning able to give rise to a single cell lineage (e.g.,spermatogenic stem cells).

Totipotent stem cell: The term refers to a cell having the potential togive rise to any and all types of human cells such as all three germlayer lineages and extraembryonic lineages. It can give rise to anentire functional organism.

In the present disclosure, any gene or protein name can refer to thegene or the protein in any species. For example, PDX1 or Pdx1 are usedinterchangeably and can refer to either murine Pdx1 or human PDX1 or toPdx1 in another species.

In the present disclosure, a “−” sign after a gene or protein name meansthat the gene or protein is not expressed, while a “+” sign after a geneor protein name means that the gene or protein is expressed. Thus PDX1−or PDX1− cells are cells that do not express PDX1, while PDX1+ or PDX1+cells are cells that express PDX1.

Generation of Beta Cells

The present methods allow generation of glucose-responsive beta cellsfrom a starting cell population comprising at least one cell capable ofdifferentiation. In one embodiment, the cell capable of differentiationis a pluripotent stem cell, such as a pluripotent stem cell, for examplea human pluripotent stem cell. In another embodiment, the cell capableof differentiation is a pancreatic progenitor cell.

The present methods provide a protocol for differentiating a cellpopulation comprising pluripotent stem cells into beta cells. This canbe done by following the following steps:

-   -   i) Incubating said cell population in RPMI medium comprising        Activin A and a glycogen synthase kinase (GSK3) inhibitor for a        duration of one day, thereby differentiating at least part of        the cell population into definitive endoderm cells;    -   ii) Incubating the cell population of i) in RPMI medium        comprising B27−insulin, for a duration of between 3 and 6 days,        thereby further differentiating the cell population into        definitive endoderm cells;    -   iii) Incubating the cell population of ii) in DMEM/F12 medium        comprising B27+insulin and retinoic acid, for a duration of        between 3 and 6 days, thereby differentiating at least part of        the cell population into gut tube cells;    -   iv) incubating the cell population of iii) in DMEM/F12 medium        comprising B27+insulin and human FGF2, and optionally human        Noggin, for a duration of between 3 and 6 days, thereby        differentiating at least part of the cell population into        posterior foregut cells;    -   v) Incubating the cell population of iv) in DMEM/F12 medium        comprising B27+insulin,        ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)        (TPB), and human Noggin for a duration of between 3 and 6 days,        thereby differentiating at least part of the cell population        into early pancreatic progenitor cells; and    -   vi) Incubating the cell population of v) in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        and human Noggin for a duration of between 3 and 6 days, thereby        differentiating at least part of the cell population into mature        pancreatic progenitor cells; and    -   vii) Further incubating the cell population of vi) for an        additional 7 to 23 days, thereby differentiating at least part        of the cell population into beta cells.

In all embodiments, the medium used in step vii) is preferably the sameas the medium used in step vi). For all steps, the medium may berefreshed every day or every second day, as is known to the skilledperson.

In some embodiments of the invention, the cell population obtained instep v) may be enriched for cells expressing PDX1 and NKX6.1 to obtainan enriched cell population. In this case, steps vi) and vii) above maybe replaced by steps viii) and ix) below:

-   -   viii) Incubating the enriched cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration of between 1 and        2 days; and    -   ix) Incubating the cell population obtained in step viii) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration of between 7 and 14 days.

In some embodiments of the invention, the cell population obtained instep vi) may be enriched for cells expressing PDX1 and NKX6.1 to obtainan enriched cell population. In this case, step vii) above may bereplaced by steps viii) and ix) below:

-   -   viii) Incubating the enriched cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration of between 1 and        2 days; and    -   ix) Incubating the cell population obtained in step viii) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration of between 7 and 14 days.

In other embodiments, the starting cell population comprising at leastone cell capable of differentiation is a population comprisingpancreatic progenitor cells expressing PDX1 and NKX6.1. In suchembodiments, the method of differentiating these cells into beta cellscomprises the steps of:

-   -   viii) Incubating the starting cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration of between 1 and        2 days; and    -   ix) Incubating the cell population obtained in step viii) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration of between 7 and 14 days.

The starting population of cells may be as described herein below. Insome embodiments, the starting cell population comprises pancreaticprogenitor cells and has been enriched for cells expressing PDX1 andNKX6.1 as described herein below, for example by taking advantage ofcell surface markers. The present methods can then be applied to such“sorted” cell populations to further differentiate them. In someembodiments, the starting cell population has not been enriched forcells expressing PDX1 and NKX6.1.

In one embodiment, the starting cell population comprises pancreaticprogenitor cells and has been enriched for cells expressing PDX1 andNKX6.1 by being contacted with a first ligand which binds to a firstmarker specific for PDX1− cells and selecting the cells that do not bindto said first ligand from said cell population.

In another embodiment, the starting cell population comprises pancreaticprogenitor cells and has been enriched for cells expressing PDX1 andNKX6.1 by being contacted with a second ligand which binds to a secondmarker specific for PDX1+ cells and selecting the cells that bind tosaid second ligand from the cells that do not bind to said secondligand, thereby enriching the cell population for PDX1+ cells.

In another embodiment, the starting cell population comprises pancreaticprogenitor cells and has been enriched for cells expressing PDX1 andNKX6.1 by being contacted with a third ligand which binds to a thirdmarker specific for PDX1+ NKX6.1+ cells and selecting the cells thatbind to said third ligand from the cells that do not bind to said thirdligand, thereby enriching the cell population for PDX1+ NKX6.1+ cells.

In another embodiment, the starting cell population comprises pancreaticprogenitor cells and has been enriched for cells expressing PDX1 andNKX6.1 by being contacted with, in either order:

-   -   a) a first ligand which binds to a first marker specific for        PDX1− cells and selecting the cells that do not bind to said        first ligand from said cell population; and    -   b) a second ligand which binds to a second marker specific for        PDX1+ cells and selecting the cells that bind to said second        ligand from the cells that do not bind to said second ligand,        thereby enriching the cell population for PDX1+ cells.

In another embodiment, the starting cell population comprises pancreaticprogenitor cells and has been enriched for cells expressing PDX1 andNKX6.1 by being contacted with, in either order:

-   -   a) a first ligand which binds to a first marker specific for        PDX1− cells and selecting the cells that do not bind to said        first ligand from said cell population; and    -   b) a third ligand which binds to a third marker specific for        PDX1+ NKX6.1+ cells and selecting the cells that bind to said        third ligand from the cells that do not bind to said third        ligand, thereby enriching the cell population for PDX1+ NKX6.1+        cells.

In another embodiment, the starting cell population comprises pancreaticprogenitor cells and has been enriched for cells expressing PDX1 andNKX6.1 by being contacted with, in either order:

-   -   a) a second ligand which binds to a second marker specific for        PDX1+ cells and selecting the cells that bind to said second        ligand from the cells that do not bind to said second ligand,        thereby enriching the cell population for PDX1+ cells; and    -   b) a third ligand which binds to a third marker specific for        PDX1+ NKX6.1+ cells and selecting the cells that bind to said        third ligand from the cells that do not bind to said third        ligand, thereby enriching the cell population for PDX1+ NKX6.1+        cells.

In another embodiment, the starting cell population comprises pancreaticprogenitor cells and has been enriched for cells expressing PDX1 andNKX6.1 by being contacted with, in either order:

-   -   a) a first ligand which binds to a first marker specific for        PDX1− cells and selecting the cells that do not bind to said        first ligand from said cell population; and    -   b) a second ligand which binds to a second marker specific for        PDX1+ cells and selecting the cells that bind to said second        ligand from the cells that do not bind to said second ligand,        thereby enriching the cell population for PDX1+ cells; and c) a        third ligand which binds to a third marker specific for PDX1+        NKX6.1+ cells and selecting the cells that bind to said third        ligand from the cells that do not bind to said third ligand,        thereby enriching the cell population for PDX1+ NKX6.1+ cells.

For any of the above embodiments, CD49d/ITGA4 is a preferred firstligand. For any of the above embodiments, FOLR1, CDH1/ECAD, F3/CD142,PDX1, FOXA2, EPCAM, HES1 and GATA4 are preferred second ligands. FOLR1is even more preferred. For any of the above embodiments, GP2, SCN9A,MPZ, NAALADL2, KCNIP1, CALB1, SOX9, NKX6.2 and NKX6.1 are preferredthird ligands. More preferred are GP2, SCN9A, MPZ, NAALADL2, KCNIP1 andCALB1. Even more preferred is GP2.

In some embodiments, the starting cell population comprises pancreaticprogenitor cells, and may have been obtained as described herein byproviding a cell population comprising pluripotent stem cells andperforming steps i) to v) above. In other embodiments, the starting cellpopulation comprises pancreatic progenitor cells, and may have beenobtained as described herein by providing a cell population comprisingpluripotent stem cells and performing steps i) to vi) above.

Starting Cell Population

In a first step, a cell population (the starting cell population)comprising at least one cell capable of differentiation is provided. Insome embodiments, the cell capable of differentiation is a pancreaticprogenitor cell expressing PDX1 and NKX6.1.

In some embodiments, the starting cell population comprises at least 5%pancreatic progenitor cells, such as at least 10% pancreatic progenitorcells, such as at least 15% pancreatic progenitor cells, such as atleast 20% pancreatic progenitor cells, such as at least 25% pancreaticprogenitor cells, such as at least 30% pancreatic progenitor cells, suchas at least 35% pancreatic progenitor cells, such as at least 40%pancreatic progenitor cells, such as at least 45% pancreatic progenitorcells, such as at least 50% pancreatic progenitor cells, such as atleast 55% pancreatic progenitor cells, such as at least 60% pancreaticprogenitor cells, such as at least 65 pancreatic progenitor cells, suchas at least 70% pancreatic progenitor cells, such as at least 75%pancreatic progenitor cells, such as at least 80% pancreatic progenitorcells, such as at least 85% pancreatic progenitor cells, such as atleast 90% pancreatic progenitor cells, such as at least 95% pancreaticprogenitor cells.

In order to determine the fraction of progenitor cells comprised in acell population, for example in the starting population, methods knownin the art can be employed, such as, but not limited to, immunostainingor flow cytometry methods.

Without being bound by theory, the percentage of pancreatic progenitorcells in the starting cell population can be estimated by the expressionof GP2. Thus in some embodiments, the starting cell population comprisesat least 5% cells expressing GP2, such as at least 10% cells expressingGP2, such as at least 15% cells expressing GP2, such as at least 20%cells expressing GP2, such as at least 25% cells expressing GP2, such asat least 30% cells expressing GP2, such as at least 35% cells expressingGP2, such as at least 40% cells expressing GP2, such as at least 45%cells expressing GP2, such as at least 50% cells expressing GP2, such asat least 55% cells expressing GP2, such as at least 60% cells expressingGP2, such as at least 65% cells expressing GP2, such as at least 70%cells expressing GP2, such as at least 75% cells expressing GP2, such asat least 80% cells expressing GP2, such as at least 85% cells expressingGP2, such as at least 90% pancreatic progenitor cells, such as at least95% pancreatic progenitor cells. GP2 expression can be determined bymethods known in the art, such as immunostaining methods, flow cytometrymethods or quantitative measurements of transcription levels.

Likewise, without being bound by theory, the percentage of PDX1+ NKX6.1+cells in the starting cell population can be estimated by the expressionof GP2. Thus in some embodiments, the starting cell population comprisesat least 5% cells expressing GP2, such as at least 10% cells expressingGP2, such as at least 15% cells expressing GP2, such as at least 20%cells expressing GP2, such as at least 25% cells expressing GP2, such asat least 30% cells expressing GP2, such as at least 35% cells expressingGP2, such as at least 40% cells expressing GP2, such as at least 45%cells expressing GP2, such as at least 50% cells expressing GP2, such asat least 55% cells expressing GP2, such as at least 60% cells expressingGP2, such as at least 65% cells expressing GP2, such as at least 70%cells expressing GP2, such as at least 75% cells expressing GP2, such asat least 80% cells expressing GP2, such as at least 85% cells expressingGP2, such as at least 90% pancreatic progenitor cells, such as at least95% pancreatic progenitor cells. GP2 expression can be determined bymethods known in the art, such as immunostaining methods, flow cytometrymethods or quantitative measurements of transcription levels.

In some embodiments, the cell population may be derived or isolated froman individual, such as, but not limited to, a mammal, for example ahuman.

In some embodiments, the cells capable of differentiation arepluripotent stem cells, for example human pluripotent stem cells(hPSCs). hPSCs include human induced pluripotent stem cells (hiPSCs),human embryonic stem cells (hESCs) and naïve human stem cells (NhSCs).

In one embodiment, the starting cell population is obtained from apancreas, including a foetal pancreas or an adult pancreas. In oneaspect, the pancreas is from a mammal, such as a human.

In another embodiment, the starting cell population is a somatic cellpopulation. In some embodiments, the starting cell population comprisesat least one pancreatic progenitor cell expressing PDX1 and NKX6.1 andis obtained from a somatic cell population. In a further aspect of theinvention, the somatic cell population has been induced tode-differentiate into an embryonic-like stem cell (ESC, e.g. apluripotent stem cell, or hESCs for human ESCs). Such dedifferentiatedcells are also termed induced pluripotent stem cells (IPSCs, or hIPSCsfor human IPSCs).

In yet another embodiment, the starting cell population is ESCs orhESCs. In one embodiment, the starting cell population is obtained fromESCs or hESCs. In some embodiments, the starting cell population is apopulation of pluripotent stem cells such as ESC like-cells.

In some embodiments, a cell population comprising at least onepancreatic progenitor cell may be obtained by methods known in the art,before steps viii) and ix) as described herein are performed. Forexample, differentiation can be induced in embryoid bodies and/or inmonolayer cell cultures or a combination thereof.

In one aspect of the invention, the starting cell population is ofmammalian origin. In one aspect of the invention, the starting cellpopulation is of human origin.

In one aspect of the invention, the starting cell population is obtainedfrom one or more donated pancreases. The methods described herein arenot dependent on the age of the donated pancreas. Accordingly,pancreatic material isolated from donors ranging in age from embryos toadults can be used.

Once a pancreas is harvested from a donor, it is typically processed toyield individual cells or small groups of cells for culturing using avariety of methods. One such method calls for the harvested pancreatictissue to be cleaned and prepared for enzymatic digestion. Enzymaticprocessing is used to digest the connective tissue so that theparenchyma of the harvested tissue is dissociated into smaller units ofpancreatic cellular material. The harvested pancreatic tissue is treatedwith one or more enzymes to separate pancreatic cellular material,substructures, and individual pancreatic cells from the overallstructure of the harvested organ. Collagenase, DNAse, lipasepreparations and other enzymes are contemplated for use with the methodsdisclosed herein.

Isolated source material can be further processed to enrich for one ormore desired cell populations prior to performing the present methods.In some aspects unfractionated pancreatic tissue, once dissociated forculture, can also be used directly in the culture methods of theinvention without further separation. However, unfractionated pancreatictissue, once dissociated for culture, can also be used directly in theculture methods of the invention without further separation, and willyield the intermediate cell population. In one embodiment the isolatedpancreatic cellular material is purified by centrifugation through adensity gradient (e. g., Nycodenz, Ficoll, or Percoll). The mixture ofcells harvested from the donor source will typically be heterogeneousand thus contain alpha cells, beta cells, delta cells, ductal cells,acinar cells, facultative progenitor cells, and other pancreatic celltypes.

A typical purification procedure results in the separation of theisolated cellular mate-rial into a number of layers or interfaces.Typically, two interfaces are formed. The upper interface isislet-enriched and typically contains 10 to 100% islet cells insuspension.

The second interface is typically a mixed population of cells containingislets, acinar, and ductal cells. The bottom layer is the pellet, whichis formed at the bottom of the gradient. This layer typically containsprimarily acinar cells, some entrapped islets, and some ductal cells.Ductal tree components can be collected separately for furthermanipulation.

The cellular constituency of the fractions selected for furthermanipulation will vary depending on which fraction of the gradient isselected and the final results of each isolation. When islet cells arethe desired cell type, a suitably enriched population of islet cellswithin an isolated fraction will contain at least 10% to 100% isletcells. Other pancreatic cell types and concentrations can also beharvested following enrichment. For example, the culture methodsdescribed herein can be used with cells isolated from the secondinterface, from the pellet, or from other fractions, depending on thepurification gradient used.

In one embodiment, intermediate pancreatic cell cultures are generatedfrom the islet-enriched (upper) fraction. Additionally, however, themore heterogeneous second interface and the bottom layer fractions thattypically contain mixed cell populations of islets, acinar, and ductalcells or ductal tree components, acinar cells, and some entrapped isletcells, respectively, can also be used in culture. While both layerscontain cells capable of giving rise to the enriched pancreaticprogenitor cell population described herein, each layer may haveparticular advantages for use with the disclosed methods.

In one embodiment, the starting cell population is a population of stemcells. In one embodiment, the starting cell population is a populationof stem cells that is obtained without the destruction of an embryo.Methods for obtaining stem cells without destroying embryos are known inthe art (Chung et al., 2008).

A protocol for obtaining pancreatic cells from stem cells is exemplifiedby, but not limited to, the protocols described in D'Amour, K. A. et al.(2006); Jiang, J. et al. (2007); and Kroon, E. et al. (2008), Rezania etal (2012, 2014), Felicia W. Pagliuca et al (2014). Pancreatic progenitorcells obtained using such protocols can be further differentiated tobeta cells using the methods disclosed herein, in particular steps viii)and ix).

A protocol for obtaining pancreatic cells from somatic cells or somaticcells induced to dedifferentiate into pluripotent stem cells such as ESlike-cells is exemplified by, but not limited to, the protocolsdescribed in Aoi, T. et al. (2008), Jiang, J. et al. (2007), Takahashi,K. et al. (2007), Takahashi and Yamanaka (2006), and Wernig, M. et al.(2007). Other protocols have been described by D'Amour, K. A. et al.(2006) or Kroon, E. et al. (2008). Pancreatic progenitor cells obtainedusing such protocols can be further differentiated to beta cells usingthe methods disclosed herein, in particular steps viii) and ix).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cellsuch as, for example, a nerve cell or a muscle cell. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

In some aspects “differentiate” or “differentiation” as used hereinrefers to a process where cells progress from an immature state to aless immature state. In another aspect “differentiate” or“differentiation” as used herein refers to a process where cellsprogress from an undifferentiated state to a differentiated state orfrom an immature state to a mature state. For example, undifferentiatedpancreatic cells may be able to proliferate and express characteristicsmarkers, like PDX1. Early undifferentiated embryonic pancreatic cellsmay be able to proliferate and express characteristics markers, likePDX1. In one embodiment mature or differentiated pancreatic cells do notproliferate and secrete high levels of pancreatic endocrine hormones. Insome embodiments mature or differentiated pancreatic cells do notproliferate and secrete high levels of pancreatic endocrine hormones ordigestive enzymes. In one embodiment, e.g., mature beta cells secreteinsulin at high levels. In some embodiments e.g., mature beta cellssecrete insulin at high levels in response to glucose. Changes in cellinteraction and maturation occur as cells lose markers ofundifferentiated cells or gain markers of differentiated cells. In oneembodiment loss or gain of a single marker can indicate that a cell has“matured or differentiated”. In some embodiments loss or gain of asingle marker can indicate that a cell has “matured or fullydifferentiated”. The term “differentiation factors” refers to a compoundadded to pancreatic cells to enhance their differentiation to matureendocrine cells also containing insulin producing beta cells. Exemplarydifferentiation factors include hepatocyte growth factor, keratinocytegrowth factor, exendin-4, basic fibroblast growth factor, insulin-likegrowth factor-I, nerve growth factor, epidermal growth factorplatelet-derived growth factor, and glucagon-like-peptide 1. In oneembodiment, differentiation of the cells comprises culturing the cellsin a medium comprising one or more differentiation factors.

In some embodiments, the cell population comprising at least onepancreatic progenitor cell is analysed to identify whether at least oneof the cells of the starting population expresses markers characteristicof the pancreatic endocrine lineage and selected from the groupconsisting of NGN3, NEUROD, ISL1, PDX1, NKX6.1, NKX2.2, MAFA, MAFB, ARX,BRN4, PAX4 and PAX6, GLUT2, INS, GCG, SST, pancreatic poly-peptide (PP).In some embodiments markers characteristic of the pancreatic endocrinelineage are selected from the group consisting of PDX1 and NKX6.1. Inone embodiment, a pancreatic endocrine cell is capable of expressing atleast one of the following hormones: insulin, glucagon, somatostatin,and PP. In some embodiments, a pancreatic endocrine cell is capable ofexpressing at least one of the following hormones: insulin, glucagon,somatostatin, PP and ghrelin. Suitable for use in the present inventionis a cell that expresses at least one of the markers characteristic ofthe pancreatic endocrine lineage. In one aspect of the presentinvention, a cell expressing markers characteristic of the pancreaticendocrine lineage is a pancreatic endocrine cell. The pancreaticendocrine cell may be a pancreatic hormone expressing cell.Alternatively, the pancreatic endocrine cell may be a pancreatic hormonesecreting cell.

In one embodiment, the pancreatic endocrine cell is a cell expressingmarkers characteristic of the beta cell lineage. A cell expressingmarkers characteristic of the beta cell lineage expresses PDX1 and mayfurther express at least one of the following transcription factors:NGN3, NKX2-2, NKX6.1, NEUROD, ISL1, FOXA2, MAFA, PAX4, and PAX6. In oneembodiment, a cell expressing markers characteristic of the beta celllineage is a beta cell. In one embodiment, the pancreatic endocrine cellis a cell expressing the marker NKX6.1. In another aspect of theinvention, the pancreatic endocrine cell is a cell expressing the markerPDX1. In a further aspect of the invention, the pancreatic endocrinecell is a cell expressing the markers NKX6.1 and PDX1.

PDX1 is homeodomain transcription factor implicated in pancreasdevelopment. Pax-4 is a beta cell specific factor and Pax-6 is apancreatic islet cell (specific) transcription factor; both areimplicated in islet development. Hnf-3 beta (also known as FoxA2)belongs to the hepatic nuclear factor family of transcription factors,which is characterized by a highly conserved DNA binding domain and twoshort carboxy-terminal domains. NeuroD is basic helix-loop-helix (bHLH)transcription factor implicated in neurogenesis. Ngn3 is a member of theneurogenin family of basic loop-helix-loop transcription factors. NKX2-2and NKX6.1 as used herein are members of the Nkx transcription factorfamily. Islet-1 or ISL-1 is a member of the LIM/homeodomain family oftranscription factors, and is expressed in the developing pancreas. MAFAis a transcription factor expressed in the pancreas, and controls theexpression of genes involved in insulin biosynthesis and secretion.NKX6.1 and PDX1 are co-expressed with PTF1a in the early pancreaticmultipotent cell that can develop into all cell types found in the adultpancreas (e.g., acinar, ductal, and endocrine cells). Within this cellpopulation cells that also transiently express NGN3 are found. Once acell expresses or has expressed NGN3 it will be part of the endocrinelineage, giving rise to endocrine cells (one type being the insulinproducing beta cell) that will later form the Islets of Langerhans. Inthe absence of NGN3 no endocrine cells form during pancreas development.As development progress NKX6.1 and PDX1 are co-expressed in the morecentral domain of the pancreas, which now becomes devoid of PTF1aexpression and the NKX6.1 and PDX1 positive cells can no longer giverise to acinar cells. Within this NKX6.1 and PDX1 positive cellpopulation a significant number of cells transiently co-express NGN3,marking them for the endocrine lineage like earlier in development.

In one embodiment, the cells comprised in the starting cell populationare derived from cells capable of differentiation. In a specificembodiment, the cells capable of differentiation are human pluripotentstem cells. In some embodiments, the cells capable of differentiationare selected from the group consisting of human iPS cells (hIPSCs),human ES cells (hESCs) and naive human stem cells (NhSCs).

The cells capable of differentiation may be derived from cells isolatedfrom an individual.

CDKN1a, also dubbed P21, and CDKN2a, also P16, are cell cycle specificgenes. CDKN1a (cyclin-dependent kinase inhibitor 1 or CDK-interactingprotein 1), is a cyclin-dependent kinase inhibitor that inhibits thecomplexes of CDK2 and CDK1. CDKN1 thus functions as a regulator of cellcycle progression at G1 and S phase. CDKN2a (cyclin-dependent kinaseinhibitor 2A, multiple tumor suppressor 1) is a tumor suppressorprotein. It plays an important role in cell cycle regulation bydecelerating cells progression from G1 phase to S phase, and thereforeacts as a tumor suppressor that is implicated in the prevention ofcancers.

The inventors have surprisingly found that inactivation of CDKN1a orCDKN2a in the starting cell population facilitates entry of the cellpopulation in a replicating state corresponding to the G2/M phase, inparticular when the starting cell population is PDX1 expressingpancreatic progenitor cells. Inactivation of CDKN1a or CDKN2a in thestarting cell population may also facilitate entry of the cellpopulation in the S phase. Thus inactivation of CDKN1a or CDKN2a may beuseful for obtaining mature beta cells from expanded pancreaticprogenitor cells.

In some embodiments, expression of CDKN1a and/or CDKN2a in the startingcell population is inactivated. In some embodiments, the starting cellpopulation is a population of pancreatic progenitor cells expressingPDX1. The starting cell population may also be any of the populationsdescribed above. The skilled person knows how to inactivate expressionof CDKN1a and/or CDKN2a. This may be done for instance by mutating ordeleting the corresponding genes, by known gene editing methods.Alternatively, silencing means may be employed such as siRNA in order toprevent expression of CDKN1a and/or CDKN2a. Alternatively, inhibitorspreventing correct function of CDKN1a and/or CDKN2a may be used.

Pancreatic Progenitor Cells

In the pancreas several different types of pancreatic cells may befound. The pancreatic cells include for example multi-potent pancreaticprogenitor cells, ductal/acinar progenitor cells, fully differentiatedacinar/exocrine cells, ductal/endocrine progenitor cells, endocrineprogenitor cells, early endocrine cells, and/or fully differentiatedendocrine cells. Pancreatic endoderm progenitor cells expressing PDX1and NKX6.1 have the capacity to differentiate into acinar cells, ductalcells or endocrine cells. The term ‘pancreatic progenitor cell’ or ‘truepancreatic progenitor’ refers herein to a cell, which is capable ofdifferentiating into all pancreatic lineages, including acinar, duct andendocrine, such as insulin-producing cells.

Pancreatic early endocrine cells are cells, which have initiatedexpression of one of the pancreatic endocrine hormones (insulin,glucagon, somatostatin and pancreatic polypeptide) but do not share allthe characteristics of fully mature pancreatic endocrine cells found inthe Islet of Langerhans in the adult pancreas. These cells may beendocrine cells which have turned off Ngn3 but do not share all thecharacteristics of fully differentiated pancreatic endocrine cells foundin the Islet of Langerhans in the adult pancreas, such as responsivenessto glucose, and are positive for one of the pancreatic endocrinehormones (insulin, glucagon, somatostatin, pancreatic polypeptide, andghrelin).

Pancreatic endocrine cells, or pancreatic hormone-producing cells, arecells capable of expressing at least one of the following hormones:insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin.

“Pancreatic fully differentiated endocrine cells” (also termed “fullydifferentiated en-docrine cells”, “pancreatic mature endocrine cells”,“pancreatic endocrine cells” or “pancreatic adult endocrine cells”) arecells, which share all the characteristics of fully differentiatedpancreatic endocrine cells found in the Islet of Langerhans in the adultpancreas.

The methods disclosed herein can be used to differentiate pancreaticprogenitor cells at the pancreatic endoderm stage into pancreatichormone-producing cells such as β-cells and/or insulin-producing cells.The insulin-producing cells may be responsive to glucose.

Cell Population Enriched in Pancreatic Progenitor Cells

In embodiments where the starting cell population comprises pancreaticprogenitor, the starting cell population may have been enriched by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to:        -   a) a first ligand which binds to a first marker specific for            PDX1− cells and selecting the cells that do not bind to said            first ligand from said cell population, thereby enriching            the cell population for PDX1+ cells;        -   and/or        -   b) a second ligand which binds to a second marker specific            for PDX1+ cells and selecting the cells that bind to said            second ligand from the cells that do not bind to said second            ligand, thereby enriching the cell population for PDX1+            cells;        -   and/or        -   c) a third ligand which binds to a third marker specific for            PDX1+ NKX6.1+ cells and selecting the cells that bind to            said third ligand from the cells that do not bind to said            third ligand, thereby enriching the cell population for            PDX1+NKX6.1+ cells;        -   thereby obtaining a starting cell population enriched for            pancreatic progenitor cells.

The cell population thus enriched can then be differentiated into betacells by a method comprising:

-   -   viii) Incubating the enriched cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration of between 1 and        2 days; and    -   ix) Incubating the cell population obtained in step viii) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration of between 7 and 14 days.

In some embodiments, the starting cell population comprising at leastone pancreatic progenitor cell expressing PDX1 and NKX6.1, is enrichedfor cells expressing PDX1 and NKX6.1 by taking advantage of surfacemarkers and ligands thereto, as described herein below. Such methods arealso described in detail in application WO 2016/170069.

Markers and Ligands

PDX1 (Pancreatic and duodenal homeobox 1), also known as insulinpromoter factor 1, is a transcription factor necessary for pancreaticdevelopment and β-cell maturation. In embryonic development, PDX1 isexpressed by a population of cells in the posterior foregut region ofthe definitive endoderm, and PDX1+epithelial cells give rise to thedeveloping pancreatic buds, and eventually, the whole of thepancreas—its exocrine, endocrine, and ductal cell populations. Pdx1 isalso necessary for β-cell maturation: developing β-cells co-expressPDX1, NKX6.1, and insulin, a process that results in the silencing ofMafB and the expression of MafA, a necessary switch in maturation ofβ-cells. PDX1+ pancreatic progenitor cells also co-express HIxb9, Hnf6,Ptf1a and NKX6.1 (homeobox protein Nkx-6.1), and these progenitor cellsform the initial pancreatic buds, which may further proliferate.Pancreatic endocrine cells express PDX1 and NKX6.1 (PDX1+ NKX6.1+cells).

The inventors have previously identified surface markers specific forcells that do not express PDX1 (PDX1− cells), while other markers wereidentified as being specific for cells that express PDX1 (PDX1+), andyet others as being specific for cells that express PDX1 and NKX6.1.Molecules capable of binding to such markers shall herein be referred toas “ligands” and can be used to isolate true pancreatic progenitorcells.

Accordingly, a starting cell population may be enriched for pancreaticprogenitor cell by a method comprising the steps of providing a cellpopulation comprising at least one pancreatic progenitor cell, whereinthe pancreatic progenitor cell expresses PDX1 and NKX6.1; and exposingsaid cell population to:

-   -   a) a first ligand which binds to a first marker specific for        PDX1− cells and selecting the cells that do not bind to said        first ligand from said cell population, thereby enriching the        cell population for PDX1+ cells;    -   and/or    -   b) a second ligand which binds to a second marker specific for        PDX1+ cells and selecting the cells that bind to said second        ligand from the cells that do not bind to said second ligand,        thereby enriching the cell population for PDX1+ cells;    -   and/or    -   c) a third ligand which binds to a third marker specific for        PDX1+ NKX6.1+ cells and selecting the cells that bind to said        third ligand from the cells that do not bind to said third        ligand, thereby enriching the cell population for PDX1+NKX6.1+        cells;    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

It will be understood that a cell population can be exposed to any ofthe first and/or second and/or third ligand in simultaneous steps or insubsequent steps and that the steps can be performed in any order. Insome embodiments, the cell population is exposed only to only one of thefirst, second or third ligand. In other embodiments, the cell populationis exposed to two or three of the first, second or third ligand. In someembodiments, the cell population is exposed to the first, the second andthe third ligand simultaneously. In some embodiments, the cellpopulation is exposed to the first ligand and to the second ligand inseparate steps. In other embodiments, the cell population is exposed tothe first ligand and to the third ligand in separate steps. In otherembodiments, the cell population is exposed to the first ligand and tothe second or third ligand simultaneously. In other embodiments, thecell population is exposed to the first ligand and in a separate step isexposed to the second and third ligand simultaneously. In otherembodiments, the cell population is exposed simultaneously to the firstand second or third ligand. In other embodiments, the cell population isexposed simultaneously to the first and second ligand, and is exposed tothe third ligand in a separate step. In other embodiments, the cellpopulation is exposed simultaneously to the first and third ligand, andis exposed to the second ligand in a separate step.

Cell populations enriched for cells expressing PDX1 and NKX6.1 using anyof these methods can be differentiated into beta cells by:

-   -   viii) Incubating the enriched cell population in DMEM/F12 medium        comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide,        human Noggin and Rock inhibitor for a duration of between 1 and        2 days; and    -   ix) Incubating the cell population obtained in step viii) in        DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5        inhibitor, Nicotinamide, human Noggin without Rock inhibitor for        a duration of between 7 and 14 days.

Ligands

After a cell population comprising at least one pancreatic progenitorcell has been provided, said population may be exposed to a first ligandwhich binds to a first marker specific for PDX1− cells and the cellsthat do not bind to said first ligand are selected. This negativeseparation results in a cell population enriched for PDX1+ cells. Thecell population is exposed to a second ligand which binds a markerspecific for PDX1+ cells and/or to a third ligand binding a third markerspecific for PDX1+ NKX6.1+ cells and the cells binding to the secondand/or third ligand are selected. It will be understood that exposure toeach of the first and second and/or third ligand can be performedsimultaneously or in separate steps.

Accordingly, in one embodiment, the cell population is exposed to afirst ligand which binds to a first marker specific for PDX1− cells.After the cells that do not bind to the first ligand have been selected,the cell population, now enriched for PDX1+ cells, is exposed to asecond ligand which binds a marker specific for PDX1+ cells and/or to athird ligand binding a third marker specific for PDX1+ NKX6.1+ cells andthe cells binding to the second and/or third ligand are selected.

In another embodiment, the cell population is exposed to the firstligand, to the second ligand and/or to the third ligand simultaneously,and the cells that do not bind the first ligand but that bind to thesecond and/or third ligand are selected.

Each of the ligands disclosed herein is a moiety that specifically bindsor cross-reacts to a marker, i.e. a marker specific for PDX1+ cells,PDX1− cells or PDX1+ NKX6.1+ cells. The term ‘ligand’ or ‘ligands’ willbe used as a generic term to refer to any of the first, second or thirdligand.

Such ligands and markers are described in more detail in application WO2016/170069, in particular in the section entitled “Ligands”.

First Ligand

The present method for enriching a cell population for pancreaticprogenitor cell comprises the steps of:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to:        -   a) a first ligand which binds to a first marker specific for            PDX1− cells and selecting the cells that do not bind to said            first ligand from said cell population, thereby enriching            the cell population for PDX1+ cells;        -   and/or        -   b) a second ligand which binds to a second marker specific            for PDX1+ cells and selecting the cells that bind to said            second ligand from the cells that do not bind to said second            ligand, thereby enriching the cell population for PDX1+            cells;        -   and/or        -   c) a third ligand which binds to a third marker specific for            PDX1+ NKX6.1+ cells and selecting the cells that bind to            said third ligand from the cells that do not bind to said            third ligand, thereby enriching the cell population for            PDX1+NKX6.1+ cells;        -   thereby obtaining a cell population enriched for pancreatic            progenitor cells.

Accordingly, after providing a starting cell population comprising atleast one pancreatic progenitor cell, wherein the pancreatic progenitorcell expresses PDX1 and NKX6.1, the cell population may be exposed to afirst ligand which binds a first marker specific for PDX1− cells and thecells that do not bind to said first ligand may be selected, therebyenriching for PDX1-expressing (PDX1+) cells.

The first ligand can be a ligand as described above. In someembodiments, the first ligand is a ligand capable of recognising andbinding a cell surface marker. In some embodiments, the cell surfacemarker is CD49d. In some embodiments, the first ligand is a monoclonalor polyclonal antibody or fragment thereof directed against CD49d. Thefirst ligand may be conjugated to a label, for example in order tofacilitate selection of the cells that do not bind to the first ligand,as detailed above.

Selection of the cells that do not bind to the first ligand may beperformed by methods known in the art such as flow cytometry.Accordingly, in some embodiments, expression of the first marker may bedetected by flow cytometry.

Second Ligand

The method may further comprise the step of exposing the startingpopulation (or the cells that do not express PDX1 after selection with afirst ligand as described above) to a second ligand which binds to asecond marker specific for PDX1+ cells and selecting the cells that bindto said second ligand from the cells that do not bind to said secondligand, thereby obtaining a cell population enriched for PDX1+ cells. Asa result, a population enriched for pancreatic progenitor is obtained.The enriched population may in particular be enriched for posteriorforegut PDX1+ cells.

The second ligand can be a ligand as described above. In someembodiments, the second ligand is a ligand capable of recognising andbinding a cell surface marker. In some embodiments, the second ligandcan recognise and bind to a second target selected from the groupconsisting of FOLR1, CDH1/ECAD, F3/CD142, PDX1, FOXA2, EPCAM, HES1, andGATA4. In some embodiments, the second ligand is a monoclonal orpolyclonal antibody or fragment thereof directed against a second targetselected from the group consisting of FOLR1, CDH1/ECAD, F3/CD142, PDX1,FOXA2, EPCAM, HES1, and GATA4. The second ligand may be conjugated to alabel, for example in order to facilitate selection of the cells thatbind to the second ligand, as detailed above. In a preferred embodiment,the second ligand is capable of recognising and binding to FOLR1.

Selection of the cells that bind to the second ligand may be performedby methods known in the art such as flow cytometry. Accordingly, in someembodiments, expression of the second marker may be detected by flowcytometry.

Exposure of the cell population to the second ligand may occur at thesame time as exposure to the first ligand and optionally to the thirdligand, or it may occur in a separate step.

Third Ligand

The method may comprise the step of exposing the cell population to athird ligand which binds to a third marker specific for PDX1+ NKX6.1+cells and the cells that bind to said third ligand are selected, inorder to obtain a cell population enriched for pancreatic progenitorcells expressing both PDX1 and NKX6.1. Exposure to the third ligand maybe performed instead of exposure to the second ligand. In someembodiments, the cell population is exposed to the first and thirdligand, where the exposure to the ligands can occur simultaneously or inseparate steps. In other embodiments, the cell population is exposed tothe first, the second and the third ligand, where the exposure to theligands can occur simultaneously or in separate steps. As a result, apopulation enriched for pancreatic progenitor cells is obtained.

The third ligand can be a ligand as described above. In someembodiments, the third ligand is a ligand capable of recognising andbinding a cell surface marker. In some embodiments, the third ligand canrecognise and bind to a third target, where the third target is selectedfrom the group consisting of GP2, SCN9A, MPZ, NAALADL2, KCNIP1, CALB1,SOX9, NKX6.2, and NKX6.1. In a preferred embodiment, the third target isGP2. In some embodiments, the third ligand is a monoclonal or polyclonalantibody or fragment thereof directed against a third target selectedfrom the group consisting of GP2, SCN9A, MPZ, NAALADL2, KCNIP1, CALB1,SOX9, NKX6.2 and NKX6.1. In a preferred embodiment, the third ligand isa monoclonal or polyclonal antibody or fragment thereof directed againstGP2. The third ligand may be conjugated to a label, for example in orderto facilitate selection of the cells that bind to the third ligand, asdetailed above.

Selection of the cells that bind to the third ligand may be performed bymethods known in the art such as flow cytometry. Accordingly, in someembodiments, expression of the third marker may be detected by flowcytometry.

Accordingly, in some embodiments, a cell population, which may beobtained by steps i) to v) or by steps i) to vi) described herein, isenriched for pancreatic progenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to:        -   a) a first ligand which binds to a first marker specific for            PDX1− cells and selecting the cells that do not bind to said            first ligand from said cell population, thereby enriching            the cell population for PDX1+ cells;        -   and/or        -   b) a second ligand which binds to a second marker specific            for PDX1+ cells and selecting the cells that bind to said            second ligand from the cells that do not bind to said second            ligand, thereby enriching the cell population for PDX1+            cells;        -   and/or        -   c) a third ligand which binds to a third marker specific for            PDX1+ NKX6.1+ cells and selecting the cells that bind to            said third ligand from the cells that do not bind to said            third ligand, thereby enriching the cell population for            PDX1+NKX6.1+ cells;    -   wherein        -   the first ligand recognises and binds to CD49d,        -   the second ligand recognises and binds to a second marker            selected from the group consisting of FOLR1, CDH1/ECAD,            F3/CD142, PDX1, FOXA2, EPCAM, HES1, and GATA4, and        -   the third ligand recognises and binds to a third marker            selected from the group consisting of GP2, SCN9A, MPZ,            NAALADL2, KCNIP1, CALB1, SOX9, NKX6.2 and NKX6.1,    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

In some embodiments, a cell population, which may be obtained by stepsi) to v) or i) to vi) described herein, is enriched for pancreaticprogenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to:        -   a) a first ligand which binds to a first marker specific for            PDX1− cells and selecting the cells that do not bind to said            first ligand from said cell population, thereby enriching            the cell population for PDX1+ cells;        -   and        -   b) a third ligand which binds to a third marker specific for            PDX1+ NKX6.1+ cells and selecting the cells that bind to            said third ligand from the cells that do not bind to said            third ligand, thereby enriching the cell population for            PDX1+NKX6.1+ cells;    -   wherein        -   the first ligand recognises and binds to CD49d, and        -   the third ligand recognises and binds to a third marker            selected from the group consisting of GP2, SCN9A, MPZ,            NAALADL2, KCNIP1, CALB1, SOX9, NKX6.2 and NKX6.1,    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

In some embodiments, a cell population, which may be obtained by stepsi) to v) or i) to vi) described herein, is enriched for pancreaticprogenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to:        -   a) a first ligand which binds to a first marker specific for            PDX1− cells and selecting the cells that do not bind to said            first ligand from said cell population, thereby enriching            the cell population for PDX1+ cells;        -   and        -   b) a second ligand which binds to a second marker specific            for PDX1+ cells and selecting the cells that bind to said            second ligand from the cells that do not bind to said second            ligand, thereby enriching the cell population for PDX1+            cells;    -   wherein        -   the first ligand recognises and binds to CD49d, and        -   the second ligand recognises and binds to a second marker            selected from the group consisting of FOLR1, CDH1/ECAD,            F3/CD142, PDX1, FOXA2, EPCAM, HES1, and GATA4,    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

In some embodiments, a cell population, which may be obtained by stepsi) to v) or i) to vi) described herein, is enriched for pancreaticprogenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to:        -   a) a first ligand which binds to a first marker specific for            PDX1− cells and selecting the cells that do not bind to said            first ligand from said cell population, thereby enriching            the cell population for PDX1+ cells;        -   b) a second ligand which binds to a second marker specific            for PDX1+ cells and selecting the cells that bind to said            second ligand from the cells that do not bind to said second            ligand, thereby enriching the cell population for PDX1+            cells;        -    and        -   c) a third ligand which binds to a third marker specific for            PDX1+ NKX6.1+ cells and selecting the cells that bind to            said third ligand from the cells that do not bind to said            third ligand, thereby enriching the cell population for            PDX1+NKX6.1+ cells;    -   wherein        -   the first ligand recognises and binds to CD49d,        -   the second ligand recognises and binds to FOLR1, and        -   the third ligand recognises and binds to GP2,    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

In some embodiments, a cell population, which may be obtained by stepsi) to v) or i) to vi) described herein, is enriched for pancreaticprogenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to:        -   a) a first ligand which binds to a first marker specific for            PDX1− cells and selecting the cells that do not bind to said            first ligand from said cell population, thereby enriching            the cell population for PDX1+ cells;        -   and        -   b) a second ligand which binds to a second marker specific            for PDX1+ cells and selecting the cells that bind to said            second ligand from the cells that do not bind to said second            ligand, thereby enriching the cell population for PDX1+            cells;    -   wherein        -   the first ligand recognises and binds to CD49d,        -   the second ligand recognises and binds to FOLR1,    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

In some embodiments, a cell population, which may be obtained by stepsi) to v) or i) to vi) described herein, is enriched for pancreaticprogenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   Exposing said cell population to a third ligand which binds to a        third marker specific for PDX1+ NKX6.1+ cells and selecting the        cells that bind to said third ligand from the cells that do not        bind to said third ligand, thereby enriching the cell population        for PDX1+ NKX6.1+ cells;    -   wherein        -   the third ligand recognises and binds to a marker selected            from the group consisting of SCN9A, MPZ, NAALADL2, KCNIP1,            CALB1, SOX9, NKX6.2 and NKX6.1,    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

In a preferred embodiment, the marker is SCN9A, MPZ, NAALADL2, KCNIP1,GP2 or CALB1. In a specific embodiment, the marker is GP2.

In some embodiments, a cell population, which may be obtained by stepsi) to v) or i) to vi) described herein, is enriched for pancreaticprogenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to a third ligand which binds to a        third marker specific for PDX1+ NKX6.1+ cells and selecting the        cells that bind to said third ligand from the cells that do not        bind to said third ligand, thereby enriching the cell population        for PDX1+ NKX6.1+ cells;    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

In some embodiments, a cell population, which may be obtained by stepsi) to v) or i) to vi) described herein, is enriched for pancreaticprogenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to a third ligand which binds to a        third marker specific for PDX1+ NKX6.1+ cells and selecting the        cells that bind to said third ligand from the cells that do not        bind to said third ligand, thereby enriching the cell population        for PDX1+ NKX6.1+ cells;    -   wherein the third ligand recognises and binds to GP2,    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

In some embodiments, a cell population, which may be obtained by stepsi) to v) or i) to vi) described herein, is enriched for pancreaticprogenitor cells by:

-   -   providing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1; and    -   exposing said cell population to:        -   a) a second ligand which binds to a second marker specific            for PDX1+ cells and selecting the cells that bind to said            second ligand from the cells that do not bind to said second            ligand, thereby enriching the cell population for PDX1+            cells;        -   and        -   b) a third ligand which binds to a third marker specific for            PDX1+ NKX6.1+ cells and selecting the cells that bind to            said third ligand from the cells that do not bind to said            third ligand, thereby enriching the cell population for            PDX1+NKX6.1+ cells;    -   wherein        -   the second ligand recognises and binds to FOLR1,        -   the third ligand recognises and binds to GP2,    -   thereby obtaining a cell population enriched for pancreatic        progenitor cells.

It will be understood that methods for generating hormone producing betacells such as insulin-producing beta cells include sorting GP2 positivecells isolated by flow cytometry or other similar methods anddifferentiating said sorted cells further using the methods describedherein.

The present methods allow differentiation of a population of at leastpart of a starting cell population comprising at least one pancreaticprogenitor cell expressing PDX1 and NKX6.1 into beta cells, preferablyglucose-responsive and/or insulin-producing beta cells. In someembodiments, the starting cell population has been enriched forpancreatic progenitor cells as described herein above.

Preferably, at least one cell of the starting cell population cells hasthe capability to differentiate further. The population may have thecapability to differentiate further into pancreatic hormone-producingcells. In some embodiments, at least one of the pancreatichormone-producing cells is an insulin-producing cell. In someembodiments, at least one of the pancreatic hormone-producing cells isresponsive to glucose. In some embodiments, at least one of thepancreatic hormone-producing cells is an insulin-producing cell, whichis also responsive to glucose. In some embodiments, at least one cell ofthe cell population enriched for pancreatic progenitor cells can produceinsulin-producing islet cells.

In some embodiments, the at least one of the pancreatichormone-producing cells has increased expression of at least one ofinsulin, C-peptide, Insm-1, 151-1, MafA and MafB compared to a cellpopulation that has been incubated in the absence of the Yap1 inhibitor.In one embodiment, the pancreatic hormone-producing cell is a mature βcell.

In some embodiments, CDKN1a and/or CDKN2a is inactivated in the startingcell population. In one embodiment, CDKN1a is inactivated. In anotherembodiment, CDKN2a is inactivated. In another embodiment, CDKN1a andCDKN2a are both inactivated. In another embodiment, CDKN1a and CDKN2 areinactivated sequentially, i.e. one of CDKN1a and CDKN2a is inactivatedin a first step, and the other of CDKN1a and CDKN2a is inactivated in asecond step; the first and second steps may overlap in time or beindependent.

Treatment of Metabolic Disorder

Also disclosed herein is a cell population comprising beta cells,obtainable any of the methods disclosed herein, for treatment of ametabolic disorder in an individual in need thereof. The beta cells arepreferably glucose-responsive and/or capable of producing a pancreatichormone such as insulin.

Also disclosed herein is a method of treatment of a metabolic disorderin an individual in need thereof, wherein the method comprises a step ofproviding a population of beta cells obtainable by the methods disclosedherein and transplanting said population of beta cells into saidindividual.

The cell populations described herein can be used for treatment ofmetabolic disorders. Glucose-responsive beta cells obtained by themethods described herein are used for treating a metabolic disorder. Thestarting cell population may have been further enriched for pancreaticprogenitors as described above, for example by the use of ligandsbinding markers as detailed above and isolation by flow cytometry. Theseisolated cells can be stored prior to use, or they can be usedimmediately. The cells may be differentiated further as describedherein. Once a cell population with the desired characteristics isobtained, the cells are transplanted into an individual in need thereof.As an example, such cell-based therapy is useful for transplantinginsulin-producing β-cells in individuals suffering from diabetes,whereby insulin production may be restored in vivo. If the starting cellpopulation is derived from the patient him/herself, the risks of adverseimmune reactions such as rejection of the transplanted cells may bereduced. As an alternative to transplanting insulin-producing β-cellsinto a patient, pancreatic progenitor cells, such as the cellsobtainable by the methods described herein, can also be transplanted.

The term ‘metabolic disorder’ as used herein shall be construed to referto endocrine, nutritional and metabolic diseases. Preferably, thedisorder is related to a pancreatic disorder. Examples of metabolicdisorders are: diabetes mellitus, including type 1 and type 2 diabetes.

Diabetes mellitus, commonly referred to as diabetes, is a group ofmetabolic diseases in which there are high blood sugar levels over aprolonged period. Several types of diabetes exist, including type 1diabetes, type 2 diabetes and gestational diabetes. Type 1 diabetes ischaracterized by loss of the insulin-producing β cells of the islets ofLangerhans in the pancreas, leading to insulin deficiency. Type 2diabetes is characterized by insulin resistance, which may be combinedwith relatively reduced insulin secretion. The defective responsivenessof body tissues to insulin is believed to involve the insulin receptor.Gestational diabetes, which resembles type 2 diabetes, occurs in about2-10% of all pregnancies. The type of diabetes can also be classified asinsulin-dependent diabetes mellitus, non-insulin dependent diabetesmellitus, malnutrition-related diabetes mellitus or unspecified diabetesmellitus.

The methods disclosed herein can be used to generate beta cells, inparticular glucose-responsive beta cells. In some embodiments, the betacells produce insulin. Accordingly, in some embodiments there isprovided a population of beta cells for treatment of a metabolicdisorder in an individual in need thereof. In some embodiments, themetabolic disorder is selected from the group consisting of diabetesmellitus such as insulin-dependent diabetes mellitus, non-insulindependent diabetes mellitus, malnutrition-related diabetes mellitus orunspecified diabetes mellitus.

In some embodiments, the cell population for treatment of a metabolicdisorder is obtained by the methods described above.

The cell population may have been further enriched for cells expressingPDX1 and NKX6.1 by any of the following steps:

-   -   exposing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1, to a third ligand which binds to a        third marker specific for PDX1+ NKX6.1+ cells and selecting the        cells that bind to said third ligand from the cells that do not        bind to said third ligand, thereby enriching the cell population        for PDX1+ NKX6.1+ cells; wherein the third ligand recognises and        binds to GP2; or    -   exposing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1, to a second ligand which binds to a        second marker specific for PDX1+ cells and selecting the cells        that bind to said second ligand from the cells that do not bind        to said second ligand, thereby enriching the cell population for        PDX1+ cells and to a third ligand which binds to a third marker        specific for PDX1+ NKX6.1+ cells and selecting the cells that        bind to said third ligand from the cells that do not bind to        said third ligand, thereby enriching the cell population for        PDX1+ NKX6.1+ cells, wherein the second ligand recognises and        binds to FOLR1, and the third ligand recognises and binds to        GP2; or    -   exposing a cell population comprising at least one pancreatic        progenitor cell, wherein the pancreatic progenitor cell        expresses PDX1 and NKX6.1, to a first ligand which binds to a        first marker specific for PDX1− cells and selecting the cells        that do not bind to said first ligand from said cell population,        thereby enriching the cell population for PDX1+ cells; and to a        second ligand which binds to a second marker specific for PDX1+        cells and selecting the cells that bind to said second ligand        from the cells that do not bind to said second ligand, thereby        enriching the cell population for PDX1+ cells; and to a third        ligand which binds to a third marker specific for PDX1+ NKX6.1+        cells and selecting the cells that bind to said third ligand        from the cells that do not bind to said third ligand, thereby        enriching the cell population for PDX1+ NKX6.1+ cells, wherein        the first ligand recognises and binds to CD49d, the second        ligand recognises and binds to FOLR1, and the third ligand        recognises and binds to GP2;    -   or any of the methods described herein elsewhere.

In one aspect is provided a method of treatment of a metabolic disorderin an individual in need thereof, wherein the method comprises a step ofproviding a beta cell population obtainable by the methods describedherein. In some embodiments, the beta cell population produces insulin.In some embodiments, the beta cell population is glucose-responsive.

In some embodiments, the present methods comprise a step oftransplanting at least part of said cell population into the individualsuffering from a metabolic disorder.

Examples

Stem cell-based therapy for type 1 diabetes would benefit fromimplementing a cell purification step at the pancreatic endoderm stage.This will increase the safety of the final cell product, allow theestablishment of an intermediate stage stem cell bank, and provide newmeans for up-scaling β-cell manufacturing. Comparative gene expressionanalysis revealed glycoprotein 2 (GP2) as a specific cell surface markerfor isolating pancreatic endoderm cells (PECs) from differentiated hESCsand human fetal pancreas. Importantly, isolated GP2⁺ PECs efficientlydifferentiated into glucose responsive insulin-producing cells in vitro.We discovered that PECs' proliferation in vitro declines due to enhancedexpression of the CDK inhibitors CDKN1A and CDKN2A. However, weidentified a time-window when reducing CDKN1A or CDKN2A expressionincreased proliferation and yield of GP2⁺ PECs. Altogether, our resultscontribute with new tools and concepts for the isolation and use of PECsas a source for safe production of hPSC-derived β-cells.

Introduction

The recent success in generating human pluripotent stem cell(hPSC)-derived glucose responsive insulin-producing cells that sharefunctional properties with normal beta cells (Pagliuca et al., 2014,Rezania et al., 2014, Russ et al., 2015), has made the implementation ofa cell-based therapy for the treatment of type 1 diabetes a tangiblereality. The number of islet cells required for disease recovery hasbeen estimated to around 300-750 million cells per patient (Bruni etal., 2014, Pagliuca et al., 2014). Thus, to be able to generate asufficient number of hPSC-derived beta cells that is useful for a largernumber of patients it will be necessary to implement expansion steps.Towards this end, expansion of either undifferentiated hPSCs (Schulz etal., 2012) or proliferative intermediate endodermal progenitors (Chenget al., 2012, Zhu et al., 2016) has been explored.

During pancreas development, multipotent pancreatic endoderm cells(PECs) with inherent proliferative capacity, co-expressing PDX1, NKX6.1,and SOX9, are responsible for the proper growth of the organ (Kopp etal., 2011, Schaffer et al., 2010, Seymour et al., 2007). The pancreaticepithelium proliferates and expands between E8.5-E11.5 in the mouse(Stanger et al., 2007) corresponding to 25-35 days post conception inhuman development (Jennings et al., 2013, Nair and Hebrok, 2015). Incontrast to more committed cells with limited to no proliferativecapacity, such as the NEUROG3 (NGN3)⁺ endocrine progenitors (Castaing etal., 2005), PECs give rise to all mature pancreatic epithelialderivatives, including acinar, ductal, and endocrine cells (Gu et al.,2002, Herrera, 2002, Kawaguchi et al., 2002).

Previous attempts have identified putative markers for hESC-derived PECs(CD142) and endocrine cells (CD200/CD318) (Kelly et al., 2011). However,more specific PEC markers remain to be identified since CD142 labelsadditional cell types (Kelly et al., 2011).

Proliferation of pancreatic progenitors (both human and mouse) can beinduced by co-culture with mesenchymal or endothelial cells (Cheng etal., 2012, Sneddon et al., 2012) or by the addition of mitogenic signalssuch as FGFs, or EGF (Bonfanti et al., 2015, Elghazi et al., 2002, Zhuet al., 2016). However, it remains unclear whether the proliferativecapacity of PECs in vitro corresponds to the self-renewal of PE thatunderlies organ growth in vivo (Stanger et al., 2007). Thus, to developnew strategies for expanding pure populations of PECs, it is necessaryto both improve methods for isolating pure populations of PECs andunderstand how PEC proliferation is regulated. In this study, weidentified glycoprotein 2 (GP2) as a specific cell surface marker forthe isolation of human PECs from differentiated hESCs and the humanfetal pancreas. Furthermore, we showed that re-plated GP2⁺ PECs retainthe capacity to differentiate with high efficiency intoglucose-responsive insulin producing beta-like cells. In addition, wediscovered that as PECs mature into PDX1+/NKX6.1^(High) cells in vitro,the expression of the negative cell cycle regulators CDKN1a (also knownas p21) and CDKN2a (also known as p16) increase. Specifically, weidentified a temporal window in which the proliferation and yield ofearly PDX1+/NKX6.1^(Low) PECs can be enhanced through reduced expressionof CDKN1A or CDKN2A. Altogether, our study provides key elements towardsa novel strategy where isolated GP2⁺ PECs can be used as a new sourcefor production of beta cells for future cell replacement therapy in type1 diabetes.

Results Comparative Gene Expression Analysis of Pancreatic and PosteriorForegut Endoderm

To define the specific gene expression signature of PECs and identifyPEC-specific cell surface markers, we first designed a strategy forgenerating putative PECs (PDX1⁺/NKX6.1⁺, protocol A, FIGS. 1A, 1B) andposterior foregut endoderm (PFG) cells (PDX1⁺/NKX6.1⁻, protocol B, FIGS.1A, 1E). Analysis of the gene expression pattern of known pancreaticendoderm markers in PDX1⁺ and PDX1⁻ cells (GFP⁺ and GFP⁻ cells using aPDX1-eGFP hESC reporter (PDXeG)) (Figure S1A-F of Ameri et al. 2017)demonstrated that PDX1, CDH1, ONECUT1, and SOX9 were all significantlyup-regulated in the GFP⁺ cells generated by both protocols (FIGS. 1C,1D, 1F and 1G). However, while protocol A generated GFP⁺ cells withsignificant PDX1, NKX6.1 and MNX1 upregulation, the GFP⁺/PDX1⁺ cellsfrom protocol B expressed lower levels of NKX6.1 and MNX1 (FIGS. 1D,1G). Immunostainings at day 17 confirmed the expression of NKX6.1, SOX9,CDH1, and HES1 in the pancreatic endoderm cells obtained with protocol A(Figure S1G of Ameri et al. 2017 and data not shown). Altogether, theseresults suggest that the GFP⁺ cells obtained with protocol A representbona fide PECs, while GFP⁺ cells obtained with protocol B correspond toPFG cells.

Identification of Novel Cell Surface Markers for Prospective Isolationof PECs

To identify PEC-specific cell surface markers, we performed microarrayanalysis to compare the gene expression pattern in PDX1⁺/NKX6.1⁺ (GFP⁺PECs), PDX1⁺/NKX6.1⁻ (GFP⁺ PFG) and PDX1⁻ (GFP⁻) cells (FIG. 2A). Onlygenes with a fold change above 1.4 (P<0.005) were selected for furtheranalysis. A total of 3403 genes (3791 probe sets) were differentiallyexpressed among the three sample groups. Hierarchical clusteringrevealed 382 genes enriched in PECs compared to PFG cells, while 698genes were enriched in the PECs compared to GFP⁻ cells. Interestingly,115 genes were specifically enriched in PECs compared to PFG and GFP⁻cells (FIG. 2B- and Table S1). Gene ontology analysis showed thatprocesses related to proliferation (e.g. cell cycle, epithelial cellproliferation, DNA replication) were significantly enriched in thePDX1⁺/NKX6.1⁺ PECs (FIG. 2C). Consistent with our initial analysis,genes that are induced early during pancreatic endoderm specificationsuch as PDX1, HHEX, GATA4 and FOXA2 were present in both PECs and PFGcells, while markers of late PECs, such as NKX6.1, SOX9, ONECUT1/2, andPRDM16 were specifically enriched in the PEC population (FIG. 2D). Ofnote, CD142 (also known as F3) and CD200, two cell surface markerspreviously shown to enrich for pancreatic endoderm cells and endocrineprogenitors (Kelly et al., 2011), were expressed in both PECs and PFGcells (FIG. 2D).

For a more in depth analysis, nine different sub-clusters were createdby hierarchical clustering. Sub-cluster 3a represents genes enriched inthe GFP⁻ cells, including the mesenchymal markers GATA2, MEIS2, TBX2,EYA1, FGFR1, HEY2, HOXA2, and VIM. Genes enriched in both PECs and PFGcells were confined to sub-cluster 6: PDX1, CDH1, GATA4, HNF1a, F3,EPCAM, FOXA2, and HES, whereas pancreatic endoderm associated genes insub-cluster 5, such as NKX6.2, SOX9, EGFR, ERBB2, and ONECUT2, wereup-regulated in PECs (FIG. 2E). Importantly, we identified cell surfacemakers that could potentially be used for the isolation of PECs.Specifically, glycoprotein 2 (zymogen granule membrane GP2) was enrichedin PDX1⁺/NKX6.1⁺ PECs (Sub-cluster 5), Folic receptor 1 (FOLR1) in allPDX1⁺ cells (Sub-cluster 6), and Integrin alpha 4 (ITGA4 or CD49d) wasenriched in GFP⁻ cells (Sub-cluster 3a) (FIG. 2E). Overall, ourexpression analysis not only reveals a new set of genes uniquelyexpressed in PECs, but also provides putative new cell surface markersfor isolation of PECs.

Functional Validation of Novel Cell Surface Markers

To validate GP2, FOLR1, and ITGA4 for the isolation of PECs, flowcytometry analysis of differentiated PDXeG cells was carried out (FIG.3A). Double staining with antibodies against GP2 and ITGA4 showed thatthe majority of the GFP⁺ cells (76%) co-expressed GP2, while 71% of theGFP⁻ cells expressed ITGA4 at day 17. Importantly, only a low fractionof the GFP⁻ cells (3%) expressed GP2 and basically none (1%) of the GFP⁺cells expressed ITGA4 (FIG. 3A). To confirm GP2's specificity inlabeling the PDX1⁺/NKX6.1⁺ cells, gene expression analysis on sortedcell fractions (ITGA4⁺/GP2⁻, ITGA4⁻/GP2⁺, and GFP⁺/GP2⁻) was performed.This analysis revealed that the pancreas associated markers PDX1,NKX6.1, MNX1, SOX9, FOXA2, and ONECUT1 were all significantly enrichedin the ITGA4⁻/GP2+ cells compared to the ITGA4⁺/GP2⁻ cells. Furthermore,while similar levels of PDX1, SOX9, FOXA2, and ONECUT1 were expressed inGFP⁺/GP2⁻ and ITGA4⁻/GP2⁺ cells, NKX6.1 and MNX1 were exclusivelyenriched in ITGA4⁻/GP2⁺ cells (FIG. 3B). As expected, both GP2 and FOLR1were enriched in the ITGA4⁻/GP2⁺ cells, whereas ITGA4 was enriched inthe ITGA4⁺/GP2⁻ cells (Figure S2A of Ameri et al. 2017). Similar resultswere obtained from the gene expression analysis performed on the cellfractions stained with FOLR1 and ITGA4, (Figure S2B,C of Ameri et al.2017). Altogether, these results suggest that GP2 and FOLR1 representspecific markers for PECs.

Next, we confirmed the cell surface markers in genetically unmodifiedhESCs under feeder-free conditions. This adaptation resulted in fewITGA4⁺ cells (FIG. 3C and Figure S2E of Ameri et al. 2017). Consistentwith the previous results, the pancreatic markers PDX1, NKX6.1, SOX9,ONECUT1, FOXA2, MNX1 were all significantly enriched in ITGA4⁻/GP2+cells in comparison to ITGA4⁺/GP2⁻ and ITGA4⁻/GP2⁻ cells (FIG. 3D). PDX1expression was still detectable in the ITGA4⁻/GP2⁻ cells; however, thesecells expressed low levels of NKX6.1 (FIG. 3D), and GP2 (Figure S2D ofAmeri et al. 2017), suggesting that these cells most likely representPDX1⁺ PFG cells. Consistently, FOLR1 was also expressed in theITGA4⁻/GP2⁻ cell fraction (Figure S2F of Ameri et al. 2017). Moreover,although pancreatic markers were enriched in the ITGA4⁻/FOLR1⁺ cells,ITGA4⁻/FOLR1⁻ cells still expressed PDX1, NKX6.1, and GP2 (Figure S2F ofAmeri et al. 2017). These data underscore that while GP2 is highlyspecific for hPSC-derived PDX1⁺/NKX6.1⁺ PECs, FOLR1 recognizes both PECsand PFG cells.

GP2 Enables Isolation of Bona Fide PECs from Human Fetal Pancreas

To corroborate the relevance of GP2 as a specific PEC marker, weexamined the expression of GP2 and ITGA4 in human fetal pancreas at 9.1weeks in development. Consistent with differentiated hESCs, GP2 andITGA4 showed no overlap in the human fetal pancreas (FIG. 3E). WhileITGA4 is expressed in the mesenchyme, GP2 is confined to the epithelium(data not shown). qPCR analysis showed that GP2⁺ cells are significantlyenriched for PDX1 and NKX6.1 (FIG. 3F). PDX1 and NKX6.1 co-expressionwas also confirmed in the GP2⁺ cells by flow cytometry (FIG. 3G).Collectively, our results demonstrate that GP2 can be utilized forisolation of PDX1⁺/NKX6.1⁺ PECs from heterogeneous populations ofdifferentiated hPSCs, as well as from human fetal pancreas in vivo.

Validation of GP2 Using an Independent Differentiation Protocol

To further substantiate the ability of GP2 to specifically recognizePECs, we used a slightly modified version of a published feeder-freedifferentiation protocol (Rezania et al., 2013) (FIG. 4A-4D). Thisprotocol generates a more heterogeneous cell population with less GP2+cells (FIG. 4B) in comparison to our modified protocol (FIGS. 4A, 5B).Consistent with the results shown above, GP2⁺/GFP⁺ cells expressed highlevels of the PEC-associated genes PDX1, NKX6.1, SOX9, and GP2 (FIG.4C). FOLR1 expression was detected in all sorted populations (GP2⁻/GFP⁻,GP2⁻/GFP⁺ and GP2⁺/GFP⁺ cells), highlighting again that GP2 is a morespecific marker for PECs compared to FOLR1 (FIG. 4C). As expected, thehighest level of ITGA4 was expressed in the GP2⁻/GFP⁻ cells (FIG. 4C).GP2-mediated enrichment of PECs was also confirmed at the protein levelby co-staining the different cell fractions with antibodies against PDX1and NKX6.1 (FIG. 4D). Finally, independent quantification analysisshowed a similar percentage of GP2⁺ and PDX1⁺/NKX6.1⁺ cells at the PEstage (14.8% GP2+ cells vs 15% PECs) (FIGS. 4E-4G). Taken together,these results unambiguously show that GP2 specifically labelsPDX1⁺/NKX6.1⁺ PECs.

Comparative Analysis of GP2 with CD142 and CD200

Analysis of the expression pattern of the previously reported cellsurface markers CD142, CD200, and CD318 (Kelly et al., 2011) revealedthat CD318 was significantly enriched in the PDX1⁻/GFP⁻ cells (data notshown), while CD142 and CD200 were present on both PDX1⁺/GFP⁺ andPDX1⁻/GFP⁻ cells (Figure S3A of Ameri et al. 2017). Comparative analysisof GP2 and CD142/CD200 stainings revealed that CD142 and CD200 labeledthe majority of the differentiated cells, while GP2 only stained asubset of the cells (Figure S3A-C of Ameri et al. 2017). qPCR analysisof the sorted cell populations showed an enrichment of the PE specificgenes PDX1, NKX6.1, and SOX9 in GP2⁺ cells compared to CD142⁺ and CD200+cells (Figure S3D of Ameri et al. 2017). Furthermore, immunostainings ofthe CD142⁺ and CD200⁺ cell fractions with PDX1 and NKX6.1 antibodiesunequivocally showed that GP2 is superior in labeling PDX1⁺/NKX6.1⁺ PECs(Figure S3E of Ameri et al. 2017, FIG. 4D). Altogether, our findingsdemonstrate that GP2 specifically labels PDX1⁺/NKX6.1⁺ PECs and can beused for purification of PECs from heterogeneous populations ofdifferentiated hPSCs independent of culture system or differentiationprotocol.

Lineage Potential of GP2⁺ PECs Towards Beta Cells

To assess the ability of isolated GP2⁺ PECs to differentiate intomono-hormonal insulin-producing beta-like cells, we optimized ourdifferentiation protocol depicted in FIG. 1B to generate glucoseresponsive beta-like cells (Protocol C, FIG. 8A). Specifically, two morestages were introduced where the cells were first differentiated in thepresence of TPB and Noggin and finally in a medium containing Forskolin,ALK5i, Noggin and Nicotinamide. This new protocol generated on average60-80% PDX1⁺/NKX6.1⁺ PECs at the PE stage (day 17 day 18) (FIG. 8B).This percentage can be directly correlated with the number of GP2^(High)cells present in the culture (FIG. 8D). Furthermore, we have observedthat the GP2^(Low) cell population shifts into a GP2^(High) cellpopulation over time (data not shown), and that this shift correlateswith the increase in NKX6.1 expression. This suggests that theGP2^(High) cells are late PECs (co-expressing PDX1 and NKX6.1) whereasGP2^(Low) cells are early PECs where NKX6.1 expression is justinitiated. As the cells are differentiated further, INS and GLU geneexpression is observed from day 23 onward (FIG. 8E). On day 32, glucoseresponsive C-peptide (CPEP⁺) cells that were also positive for PDX1 andfor NKX6.1 were detected, while very few Glucagon (GLU⁺) cells (3.6%)were observed (FIGS. 8F-8H and see also FIG. 5G).

GP2⁺ PECs sorted on day 18 were re-plated in the same differentiationmedium for two weeks (FIGS. 5A, 5B). Negative selection with ITGA4 wasnot necessary as extremely few ITGA4+ cells appeared (FIG. 5C). WhileCPEP⁺ cells emerged from both GP2^(High) cells and GP2^(Low) cells,there was a significant enrichment of CPEP⁺ cells from the GP2^(High)cells (44% from GP2^(High) vs 18% from GP2^(Low)) (FIGS. 5D, 5E).Similar to the unsorted cultures, few GLU⁺ cells were observed, althoughGP2⁺ purification at the PE stage also resulted in an enrichment of GLU⁺cells (8.3% vs 3.2%) (FIG. 5G). Furthermore, the majority of themono-hormonal CPEP⁺ cells co-expressed PDX1 and CPEP⁺/NKX6.1⁺ cells werealso observed (FIG. 5F). Importantly, insulin secretion analysis of theCPEP⁺ cells derived from GP2^(High) cells revealed an approximately2-fold increase in insulin release in response to high versus lowglucose (FIG. 5H). This result corresponds to the behavior of CPEP⁺cells derived in unsorted cultures (FIG. 8H). The level of glucoseresponsiveness is also comparable to what has been previously published(Pagliuca et al., 2014, Rezania et al., 2014). Thus, we have developedfor the first time an experimental system for generatingglucose-responsive mono-hormonal CPEP⁺ cells from isolated hPSC-derivedGP2⁺ PECs.

These experiments were finally repeated on the GMP graded hESC lineMShef-7 (FIGS. 6A-6H). Similar to the HUES4 cell line, INS and GLUexpression was detected from day 23 and onwards (FIG. 6A) and ITGA4⁺cells were scarce at day 17 (FIG. 6B).

Generation of CPEP⁺ cells was in general less efficient in MShef-7cultures compared to HUES4 (FIG. 6C). However, sorted and re-platedGP2^(High) MShef-7 cells generated significantly higher numbers of CPEP⁺cells compared to unsorted and GP2^(Low) cells (FIGS. 6D-6F, FigureS5C,D of Ameri et al. 2017). Slightly more GLU⁺ cells were observed withthe MShef-7 cell line in comparison to the HUES4 cell line (5.0% vs3.2%), and analogous to the HUES4 cultures, GP2⁺ purification resultedin an enrichment of GLU⁺ cells (11.1% vs 8.3%) (FIG. 6G). Similarly, themajority of the CPEP⁺ cells were mono-hormonal and PDX1 andCPEP⁺/NKX6.1⁺ co-expressing cells were also observed (Figure S5D, 6E ofAmeri et al. 2017). Importantly, the CPEP⁺ cells derived from theGP2^(High) cells were also glucose responsive (FIG. 6H). Altogether,these results substantiate the use of GP2 in isolating PECs with thecapacity to differentiate into beta-like cells.

Silencing of CDKN1A or CDKN2A Promotes Cell Cycle Progression of GP2⁺PECs

Current differentiation protocols of insulin-producing beta-like cellsfrom hPSCs do not support significant expansion of PECs, suggesting thatPEC proliferation is inhibited in vitro. Indeed, directeddifferentiation of hESCs towards pancreatic endoderm is associated witha decrease in proliferation (Figure S6A of Ameri et al. 2017). WhileMK167 expression is maintained until day 11, it drops concomitant withincreased expression of PDX1 and NKX6.1 (Figure S6A,B of Ameri et al.2017). Consistently, microarray analysis revealed that the negative cellcycle regulators CDKN1A (p21) and CDKN2A (p16) were specificallyenriched in the PDX1⁺/NKX6.1⁺ PECs at day 17 (Figure S6D of Ameri et al.2017). Further analysis revealed that the expression of both CDKN1A andCDKN2A increased at day 14 and remained high during subsequentdifferentiation stages (Figure S6A of Ameri et al. 2017). Both CDKN1Aand CDKN2A block cell cycle progression by inhibiting the activity ofthe cyclin/CDK complexes that regulate progression through the cellcycle (Besson et al., 2008) (Figure S6C of Ameri et al. 2017). To testwhether increased expression of CDKN1A and CDKN2A were responsible forthe drop in PEC proliferation, differentiated hESCs corresponding toPDX1⁺/NKX6.1⁺ late PECs (day 17) were re-seeded, and transfected withsiRNA against CDKN1A or CDKN2A. Knockdown efficiency was assessed byqPCR analysis 24 hours after the transfection (Figure S6E,F of Ameri etal. 2017). Unexpectedly, knocking down either CDKN1A nor CDKN2A had nosignificant impact on EdU incorporation (Figure S6G-I of Ameri et al.2017) and MKI67 expression (Figure S6J,K of Ameri et al. 2017). We alsoconfirmed that down-regulation of CDKN1A or CDKN2A expression had nonegative influence on the differentiation of the PECs, as PDX1 andNKX6.1 expression was comparable to scrambled controls (Figure S6L ofAmeri et al. 2017).

To examine if blocking the increased expression of CDKN1A and CDKN2A atan earlier time-point would increase PEC proliferation, we repeated theknockdown experiments at day 11. Knockdown efficiency was confirmed byqPCR and western blot analysis 24 hours after transfection (Figure S7A-Cof Ameri et al. 2017). In contrast to experiments performed at day 17,this time we observed that reduced expression of CDKN1A and CDKN2Aresulted in increased number of cells in the G2/M and S phases of thecell cycle, respectively (FIGS. 7A-7C). Interestingly, qPCR analysisconfirmed that MK167 expression increased 24 h after knockdown of CDKN1Abut not CDKN2A (FIGS. 7D, 7J). Nevertheless, we observed a significantincrease in the number of MKI67⁺ cells (FIGS. 6E-6F, 6K-6L) as well asin the number of PDX1⁺/NKX6.1⁺ PECs 72 h after transfection (FigureS7D,E of Ameri et al. 2017). This increase also correlated with anincrease in the total number of cells (Figure S7F of Ameri et al. 2017).Altogether, these results suggest that preventing increased expressionof CDKN1A or CDKN2A in early hESC-derived PDX1⁺/NKX6.1^(Low) PECsenhances their proliferative capacity.

To address whether the CDK inhibitors autonomously affect PECproliferation, we knocked down the expression of CDKN1A and CDKN2A andsubsequently assessed the outcome on the proliferative capacity of GP2⁺PECs specifically. Consistent with the results from the unsorted cellpopulation, knockdown of CDKN1A and CDKN2A increased the number of GP2⁺PECs that transitioned into the G2/M and S phases of the cell cycle,respectively (Figure S7G-K of Ameri et al. 2017). In sum, by preventingincreased expression of CDKN1A and CDKN2A in early hPSC-derived PECs,the proliferative capacity of PECs can be enhanced during in vitrodifferentiation (FIG. 7M).

Discussion

In this study, we report the identification of a novel cell surfacemarker, GP2, for efficient purification of human PDX1⁺/NKX6.1⁺ PECsendowed with the capacity to give rise to glucose-responsiveinsulin-producing beta-like cells. Furthermore, by counteracting theincreased expression of the cell cycle inhibitors CDKN1A and CDKN2A inthe early PECs, the proliferative capacity of hPSC-derived PECs can besustained in vitro.

The unique experimental design to compare the gene expression pattern inisolated PFG and PE cells allowed us for the first time to identify 115genes exclusively enriched within human PECs (Table S1 of Ameri et al.2017). Comparing our PE gene list with a recent study, whichsystematically analyzed genes expressed in heterogeneous cellpopulations at intermediate pancreatic differentiation stages (Xie etal., 2013), showed that 16 (including GP2) of our 115 genes overlappedwith their “PE genes” (Table S2 of Ameri et al. 2017). This new genesignature of human PE provides a unique source for interrogatingunanswered questions in PE biology, such as the molecular machineryinvolved in PEC maturation (increased expression of NKX6.1) andself-renewal.

Our genome wide expression analysis showed enrichment of the integralmembrane protein GP2 in the PDX1⁺/NKX6.1⁺ PECs. GP2 expression haspreviously been described in the acinar cells in the human adultpancreas (Hoops and Rindler, 1991, Yu et al., 2004)(http://www.proteinatlas.org/) but the role and function of GP2 duringpancreas development has not been examined. Hence, this is the firstreport showing that GP2 is expressed in the human PECs and that it canbe used as a cell surface marker for isolation of PECs. Furthermore, acomparison between GP2 and the previously published markers CD142 andCD200 (Kelly et al., 2011), demonstrated the superiority of GP2 inlabeling PDX1⁺/NKX6.1⁺ PECs both in heterogeneous populations ofdifferentiated hESCs as well as in the human fetal pancreas. Inaddition, the broad applicability of GP2 as a cell surface marker forisolation of PECs was proven by using independent differentiationprotocols and cell lines.

During development, proliferation of pancreatic progenitor cells ispromoted by factors secreted by the surrounding mesenchymal tissue(Attali et al., 2007, Bhushan et al., 2001, Ye et al., 2005). Co-cultureof pancreatic endoderm and mesenchymal cells promote expansion of thePDX1⁺ population while maintaining its progenitor identity. Theseactivities are in part mediated by FGF10 and EGF signaling (Attali etal., 2007, Bonfanti et al., 2015, Guo et al., 2013, Zhang et al., 2009).However, the underlying mechanism for how these factors promotepancreatic progenitor proliferation has not been elucidated. Here, weidentify the cell cycle inhibitors CDKN1A and CDKN2A as relevantregulators of PECs proliferation during in vitro differentiation. Weshow that increased expression of PDX1 and NKX6.1, a hallmark of latePECs, coincides with increased expression of CDKN1A and CDKN2A and asignificant decrease in the proliferative capacity of PECs. Moreover,our observation that lowered expression of CDKN1A and CDKN2A sustainsproliferation of early PECs are consistent with previous work linkingrepression of CDKN1A and CDKN2A activities to self-renewal and expansionof other stem cell or progenitor populations (Kippin et al., 2005, Koikeet al., 2014, Orford and Scadden, 2008).

Although reduction of both CDKN1A and CDKN2A levels promotes an overallincrease in proliferation of early PECs, their effect on cell cycleprogression as well as the immediate impact on MKI67 expression differs,suggesting different mechanisms of action. CDKN1A and CDKN2A belong todifferent families of CDK inhibitors. CDKN1A is a member of the Cip/Kipfamily and binds to multiple Cdk-cyclin complexes, inhibiting theircatalytic activities at the G₁/S- and G₂/M-phase checkpoints. CDKN2Abelongs to the INK4 family and blocks entry into the S phase bytargeting the CDK4/6-cyclin complexes that are present in G1 phase(Besson et al., 2008, Donovan and Slingerland, 2000, Yoon et al., 2012)(Figure S6C of Ameri et al. 2017). It is possible that the activation ofa broader range of Cdk-cyclin complexes upon reduction of CDKN1A levelsresults in a faster progression through the cell cycle compared to theCDKN2A knockdown. This may explain the observed differences in thenumber of cells in the G2/M- and S-phase. This notion could also explainthe lack of immediate transcriptional effect on MKI67 upon reducedCDKN2A levels, compared to CDKN1A. Still, as knocking down theexpression of either CDKN1A or CDKN2A promotes proliferation of PECs,they both remain relevant targets for future in vitro expansion of PECs.

Interestingly, we observed that only when the expression of CDKN1A andCDKN2A was decreased in early PECs, proliferation was restored. Previousstudies have shown that Neurog3 controls cell cycle exit in mouseendocrine progenitors at least in part through regulation of CDKN1Aexpression (Miyatsuka et al., 2011, Piccand et al., 2014). Time courseanalysis of differentiated hESCs indicates that NEUROG3 transcription isinitiated in the late PECs (data not shown), suggesting that NEUROG3 maybe responsible for the sustained expression of at least CDKN1A in thelate PECs. However, since knocking down the expression of CDKN1A andCDKN2A in late PECs is not sufficient to reinstate the proliferativecapacity of these cells, additional modulators downstream of NEUROG3must be involved in regulating proliferation and cell cycle exit in latePECs.

Future clinical trials aiming to test the safety and efficacy ofhPSCs-derived beta cells in type 1 diabetes will profit fromimplementing new cost-effective strategies for cell purification. Weenvision that using isolated GP2⁺ PECs for derivation of insulinproducing cells for clinical use will significantly improve the safetyof the final product. Furthermore, GP2⁺ PECs can be used to establish anintermediate-stage stem cell bank, permitting the use of more mature yetproliferative cells as a source of functional beta cells. Thus, futurestudies will need to focus on identifying conditions for in vitroexpansion of GP2⁺ PECs. We foresee a strategy that combinespharmacological targeting of the underlying machinery that regulatesproliferation through CDKN1A and/or CDKN2A with growth promotingsignals, such as FGFs and EGF. Once this has been achieved additionalexperiments will be required to characterize the maintenance of the PECphenotype, as well as the capacity to differentiate into functional betacells over sequential passages.

Experimental Procedures Cell Culture and Differentiation

The PDXeG clone 170-3 was maintained on MEFs in medium containingKO-DMEM, 10% knockout serum replacement (Ko-SR), 10 ng/ml bFGF, 1%non-essential amino acids (NEAA), 1% Glutamax, and beta-Mercaptoethanol(all reagents from Life Technologies). HUES4 and the PDXeG clone 170-3were adapted and maintained in DEF™-CS (Takara) whereas MShef-7 wasmaintained on laminin-521 (LN521, Biolamina) in Nutristem hESC XF medium(Biological Industries). Detailed information regarding thedifferentiation protocols can be found in the Supplemental ExperimentalProcedures.

RNA Extraction and Real-Time qPCR

Total RNA was extracted with GenElute Mammalian total RNA kit(Sigma-Aldrich). Reverse transcription was performed with SuperScriptIII, according to the manufacturer's instructions, using 2.5 μM randomhexamer and 2.5 μM oligo(dT) (Invitrogen). Real-time PCR measurementswere performed using the StepOnePlus™ system (Applied Biosystems) withSuperMix-UDG w/ROX, 400 nM of each primer, 0.125×SYBR Green 1 (allreagents from Life Technologies), with the exception of the qPCR data inFIGS. 5A-5H and 6A-6H which was generated using the LightCycler 48011(Roche) with PowerSYBR Green PCR Master Mix (AppliedBiosystems) and 500nM of each primer. Primer sequences are available as supplementary data(Table S3 of Ameri et al. 2017) and in our previous publication (Ameriet al., 2010). The data is shown as mean expression+/−standard error ofthe mean (SEM). Relative gene expression was determined using ACTB orGAPDH expression as housekeeping genes. When indicated the controlsample was arbitrarily set to a value of one in the graphs representingthe fold increase in comparison to the control sample.

Microarray Analysis of PDXeG Sorted Populations

Four replicates for each sample were collected by FACS. RNA isolationwas performed with the GenElute Mammalian total RNA kit (Sigma-Aldrich).cDNA was synthesized and amplified using Ovation RNA amplificationsystem (NuGEN) according to manufactures instructions. The labeledsamples were hybridized to the Human Gene 1.0 ST GeneChip array(Affymetrix, Santa Clara, Calif., USA). The arrays were washed, stainedwith phycoerytrin conjugated streptavidin (SAPE) using the AffymetrixFluidics Station® 450, and scanned in the Affymetrix GeneArray® 3000 7Gscanner to generate fluorescent images, as described in the AffymetrixGeneChip® protocol. Cell intensity files (CEL files) were generated inthe GeneChip® Command Console® Software (AGCC) (Affymetrix, Santa Clara,Calif., USA). Additional information can be found in the SupplementalExperimental Procedures.

Glucose-Stimulated Insulin Secretion (GSIS) Assay

Late stage cultures of differentiated hESCs were washed twice withKrebs-Ringer Bicarbonate buffer (KRB) containing 2 mM glucose. Sampleswere incubated for two hours in 2 mM glucose containing KRB to allowequilibration of cells. Fresh KRB containing 2 mM glucose was added andcells were incubated for 30 minutes, medium was collected, cells werewashed and incubated for 30 minutes in KRB containing 25 mM glucose.Medium was collected, cells were washed again and incubated with finalKRB containing 2 mM glucose and 25 mM KCl. All samples were analyzed forhuman C-peptide content using a commercially available kit fromMercodia.

siRNA Knockdown in Differentiated hESCs

Differentiated hESCs corresponding to day 11 or day 17 were dissociatedand transfected with 40 nM CDKN1A, CDKN2A, or scrambled siRNA control(Silencer Select siRNA, ThermoFisher Scientific) using LipofectamineRNAiMAX (TermoFisher Scientific). 24 hr after transfection cells wereharvested for qPCR and 72 h later cells were harvested forimmunostainings, western blot analysis and/or treated with EDU for cellcycle analysis. Immunofluorescence stainings were analyzed with LeicaAF6000 epifluorescence widefield screening microscope.

Cell Cycle Analysis by Flow Cytometry

For cell cycle analysis with flow cytometry, cells were incubated withEdU (5-Ethynyl-2′-deoxyUridine) at a concentration of 10 μM for fourhours before dissociation. Collected samples were live-stained with GP2and fixed with 4% PFA. EdU was revealed by the Click-it EdU Alexa 647Flow Cytometry Assay kit (Invitrogen). Compatible PI staining was addedto visualize the cell cycle profile based on DNA content. Analysis wasperformed using BD LSR Fortessa (BD Biosciences). 10,000 events wererecorded and doublets were excluded.

Data Analysis and Statistics

Fiji (ImageJ) software was used for all quantifications. The percentageof CPEP+ and GLU+ cells was calculated by measuring area of CPEP or GLUover DAPI area. The percentage of MKI67+ cells was calculated bymeasuring the area of MKI67/area of PDX1. The total area was estimatedby PDX1 antibody staining and DAPI. The percentage of PECs wasquantified by measuring the area of NKX6.1 over PDX1 area. 20-25randomly selected fields were chosen for each parameter. All data werestatistically analyzed by unpaired or paired Student's t-test or bymultivariate comparison (one-way ANOVA) with Bonferroni correction usingGraphPad Prism 6 Software (GraphPad Software, USA). All values aredepicted as mean±standard error of the mean (SEM) and consideredsignificant if p<0.05.

Human ES cell culture and differentiation Undifferentiated HUES4,obtained from D. A. Melton, Howard Hughes Medical Institute (HarvardUniversity, Cambridge, Mass.) were maintained on irradiated mouseembryonic fibroblasts (MEFs) (derived by Lund Transgenic Core Facility,Lund University and the Transgenic Mice Core Facility, University ofCopenhagen) in medium containing KO-DMEM, 10% knockout serum replacement(Ko-SR), 1% non-essential amino acids (NEAA), 1% Glutamax, andbeta-Mercaptoethanol (Life Technologies) and 10 ng/ml bFGF (Peprotech).Cells were passaged with Accutase (Life Technologies) and re-plated at asplit-ratio between 1:3 and 1:4. For feeder free culture, HUES4 and thePDXeG clone 170-3 were adapted and maintained in DEF™-CS (Takara) andpassaged with TrypLE E (Life Technologies). Undifferentiated MShef-7cells obtained from the Centre for Stem Cell Biology, University ofSheffield, were maintained on laminin-521 (LN521, Biolamina) inNutriStem hESC XF medium (Biological Industries). Cells were passagedwith dissociation buffer (0.5 mM EDTA). Karyotyping was performed bystandard G-banding, and for each analysis 20-25 metaphases wereevaluated (Institute for Clinical Genetics at the Universities of Lund,Sweden and Cell Guidance Systems, Cambridge, UK).

Differentiation Protocol A and B

hESCs cultured on MEFs were grown until 90% confluency anddifferentiated into definitive endoderm (DE) by using a modified versionof the D'Amour protocol (D'Amour et al., 2005). The first day ofdifferentiation, hESCs were cultured in the presence of 100 ng/ml humanActivin A (Peprotech) and 25 ng/ml Wnt3a (R&D systems) in RPMI medium(Life Technologies). The four following days 100 ng/ml human Activin Awas added together with 1× B27−insulin in RPMI medium (LifeTechnologies). To generate PECs, DE cells were first treated with 2 μMretinoic acid (RA, Sigma Aldrich) in DMEM/F12 medium containing 1×B27+insulin (Life Technologies) for 3 days and then finally treated with64 ng/ml FGF2 in combination with 100 ng/ml Noggin (Peprotech) for theremaining 9 days. To generate PFG cells, DE cells were treated with 64ng/ml FGF2 in DMEM/F12 medium containing 1× B27+insulin (LifeTechnologies) for 12 days.

Differentiation Protocol C

Feeder-free HUES4 and MShef-7 cells were differentiated in RPMI mediumcontaining 100 ng/ml Activin A (Peprotech) and 3 μM CHIR99021(SMS-Gruppen) the first day, and then with 100 ng/ml human Activin Atogether with B27-Insulin for the remaining 4 days. At day 5 and thefollowing 3 days, 2 μM retinoic acid was added in DMEM/F12 mediumcontaining 1× B27+insulin. At day eight, cells were washed, and humanFGF2 was added (64 ng/ml) on occasions together with human Noggin (50ng/ml, Peprotech) in a DMEM/F12 medium supplemented with 1× B27+insulin.Medium was changed on a daily basis throughout the protocol. To promotedifferentiation to insulin producing cells, the cells at day 11 weretreated with TPB (0.5 μM, Millipore) and Noggin (100 ng/ml) for 3 daysand for the remaining days (up to day 32), cells were treated withForskolin (10 μM, Sigma Aldrich), Alk5 inhibitor (4.5 μM, Santa Cruz),Nicotinamide (10 mM, Sigma Aldrich), Noggin (100 ng/ml) in DMEM/F-12medium containing B27 Supplement (1×). The medium was replaced everysecond day from day 17 and onwards.

Modified Rezania Protocol

The PDXeG clone 170-3 adapted to feeder-free conditions wasdifferentiated into pancreatic endoderm following Rezania et al 2013(Rezania et al., 2013) with slight modifications. Definitive endodermwas induced according to Funa et al 2015 (Funa et al., 2015). The cellswere then cultured for 2 days with 50 ng/ml FGF7 (Peprotech) in DMEM/F12containing 1× B27⁺insulin, 2 g/l sodium bicarbonate (Sigma) and 0.25 mMvitamin C (Sigma). The cells were then incubated with 2 ng/ml FGF7, 0.25μM SANT1 (Sigma), 2 μM retinoic acid (RA, Sigma Aldrich) and 100 ng/mlNoggin in DMEM high glucose (Life Technologies) supplemented with 1×B27+insulin, 2 g/l sodium bicarbonate and 0.25 mM vitamin C for 4 days.Finally, the cells were incubated with 100 ng/ml Noggin and 500 nM TPBin DMEM high glucose (Life Technologies) supplemented with 1×B27+insulin, 2 g/l sodium bicarbonate and 0.25 mM vitamin C for 3-5days.

Generation of a hESC-Derived PDX1-eGFP (PDXeG) Reporter Cell Line

The PDX1-targeting vector was constructed by inserting aneGFP-pSV40-Neon reporter cassette upstream of the PDX1 start codon(ATG), resulting in an GFP-tagged PDX1 allele. The GFP cassette wasflanked by a 12.5 kb 5′homologous arm and by a 3.5 kb 3′homologous arm.The bacterial artificial chromosome (BAC) containing the human PDX1locus (CTD-2270K21) purchased from Life Technologies was verified byrestriction enzyme digestion and sequencing. The final targetingconstruct was verified by PCR, restriction analysis and sequencing. Thecloning of the GFP-cassette, targeting and drug selection was performedas previously published (Fischer et al., 2010). After approximately 2weeks emerging clones were picked, expanded and analyzed by PCR. TheNeomycin cassette was deleted by using a CRE expression vector(NLS-CRE-IRES-Puro) that was transiently co-transfected with a DsRED orGFP plasmid into selected targeted cloned. 24 hours postelectroporation, GFP/DsRED positive single cells were FACS sorted andplated into 96-well plates. Clones were expanded and characterized forNeo excision by PCR.

Copy Number Determination

The copy number of the PDX1 targeting vector in PDXeG cells wasdetermined by qPCR as previously described (Hoebeeck et al., 2005). Afragment of the PDX1 proximal promoter was amplified and quantifiedrelative to the copy number of the single-copy reference genes ZNF80 andGPR15. Copy numbers of the PDX1 promoter fragment (pPDX1) was calibratedby comparing to untransfected hESCs (control) and normalized to thegeometric mean of two reference genes (ZNF80 and GPR15), using theamplification efficiency adjusted ΔΔCt method as previously described(Fischer et al., 2010). Measurements on all samples were performed inquadruple Statistical significance of different pPDX1 quantities betweensamples was analyzed with one-way ANOVA.

Differential Expression Analysis

The raw data (CEL files) were imported into the R where they werenormalized using Robust MultiChip Average (RMA) using quantilenormalization and Median Polish summarization. Class comparison of theexpression profiles of the three conditions, PEC-GFP-plus,PEC-GFP-minus, and PFG-GFP-plus was conducted and probe sets weredefined as being differentially expressed when they were selected in thepaired t-test, having p-values below 0.005 and a fold-change above 1.4.All samples are MIAME compliant and were handled according to SOP in themicroarray Center (http://www.rhmicroarray.com). The 12 arrays weresubmitted to ArrayExpress at EMBL using MIAMExpress. The experimentaccession number is E-MTAB-5088.

Intersections of the gene lists generated in the three comparisons;PEC-GFP-plus versus PEC-GPF-minus, PEC-GFP-plus versus PFG-GFP-plus, andPEC-GFP-minus versus PFG-GFP-plus were visualized in a Venn diagram. Theexpression pattern of the 3791 unique probe sets in the unions set ofthe three comparisons was visualized in a hierarchical clustering usingEuclidean distance and average linkage.

Gene Ontology Enrichment Analysis

The 115 gene list was used to interrogate the MSigDB for overlaps usingthe Biological Process gene sets group (C5 gene set) in the MSigD(http://www.broadinstitues.org/gsea/msigdb/annotate.jsp). Enrichedfunctions were defined by a FDR, q-value below 0.05. In the cases whereseveral biological processes represented the same biological function,the −log q value is mean of the −log q of the grouped processes. The bargraph shows the level of enrichment by −log q.

Immunohistochemical Analysis of hESCs

hESCs were fixed in 4% paraformaldehyde for 15 minutes at roomtemperature and washed three times in PBS. Fixed cells werepermeabilized with 0.5% Triton X-100 in PBS for 15 minutes and blockedin PBS-T (0.1% Triton X-100 in PBS) supplemented with 5% normal donkeyserum (Jackson Immunoresearch) for 1 h at room temperature beforeovernight incubation (at 4° C.) with the following primary antibodies:goat anti-PDX1 (R&D systems, 1:500), mouse anti-NKX6.1 (DSHB, 1:200)rabbit anti-SOX-9 (Chemicon, 1:500), goat anti-GFP (Abcam, 1:500),rabbit anti-GFP (Abcam, 1:1000), rabbit anti-MKI67 (Abcam; 1:1000), ratanti-C-PEPTIDE (DSHB, 1:1000), Guinea Pig anti-INSULIN (Daco, 1:1000),Guinea Pig anti-GLUCAGON (Linco Research, 1:1000). After overnightincubation cells were washed three times for 10 minutes in PBS, andincubated with corresponding fluorescent secondary antibodies (Alexa 488and 647, and Cy3; Jackson Immunoresearch and Invitrogen; 1:500-1:1000)for 60 min in blocking buffer at room temperature. Cell nuclei werevisualized with DAPI (Sigma-Aldrich; 1:10000). Immunofluorescencestainings were detected and analyzed on a Zeiss Axioplan 2 or with aZeiss LSM780 confocal microscope.

FACS Sorting and Re-Plating of Differentiated hESCs Differentiated cellswere dissociated with Accutase for 10-15 min at 37° C. The cells werethen washed twice in FACS buffer (PBS, 0.5% BSA). For cell sorting, GFP+and GFP− hESCs were isolated using a DiVa flow cytometer with DiVasoftware (BD Biosciences). Re-analysis after cell sorting confirmed thatthe purity of the sorted populations was >95%. Approximately150000-500000 cells were sorted from each subpopulation and used formRNA extraction. For stainings, the cell pellet was resuspended in FACSbuffer and then incubated with following antibodies (20u1antibody/million cells) for 1 h on ice: mouse anti-GP2-PE (NordicBiosite), mouse anti-ITGA4-PE (BD Biosciences), mouse anti-ITGA4-APC (BDBiosciences), mouse anti-FOLR1-APC (R&D systems). 7-Aminoactinomycin D(7AAD) or DAPI was used to remove dead cells. Stained cells wereanalyzed and sorted using a BD FACS Aria III cell sorter (BDBiosciences). For re-plating experiments, cells were sorted in PBScontaining 0.5% BSA and then gently pelleted and re-suspended indifferentiation medium supplemented with 10-15 μM Y27632.Flow Cytometry and qPCR Analysis of Human Fetal Pancreas

Human fetal pancreases from 9.1 weeks of development were dissected anddissociated into single cell suspension by using collagenase V (0.5mg/ml, SIGMA) and trypsin-EDTA 0.05% (Gibco). Single cell suspensionswere incubated with the following human antibodies: anti-CD45(Biolegend, clone H130), anti-CD31 (Biolegend, clone WM59), anti-GP2(CliniSciences, clone D277-5) and anti-ITGA4 (Biolegend, clone 9F10) for20 minutes in HBSS supplemented with 3% of fetal calf serum (FACSmedium). After incubation, cells were washed and re-suspended in FACSmedium with Propidium Iodide (0.5 mg/ml). Cells were sorted with a FACSAria III from BD Bioscience. cDNA was isolated using the CellsDirect kitfrom Invitrogen. Cells were sorted directly into PCR tubes containingcells direct 2× reaction buffer (5 μl/tube), 0.2× Assay mix (containingTaqman primers diluted (1/100) into TE buffer (0.1 mM EDTA+10 mM Tris inwater), 2.5 μl/tube), SuperScript™ RT/PlatiniumR TaqMix (0.2 μl/tube)and TE buffer (1.3 μl/tube). A minimum of 50 cells were sorted per tubeand the sorted cells were stored at −80° C. Prior to the qPCR analysis,RT and the pre-amplification were done using the thermo-cycler (GeneAmpPCR System 9700 Applied Biosystems). qPCR analysis was performed usingTaqMan primers: PDX1 (Hs00236830_m1), NKX6.1 (hs00232355_m1) and PP/A(Hs04194521_s1) and TaqMan Universal Master Mix (Applied Biosystem).Relative gene expression was determined using PP/A as a housekeepinggene.

Western Blot Analysis

Cells were lysed in RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5mM EDTA pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containingcomplete protease inhibitor cocktail (Roche). Cell lysates were resolvedon 4-12% SDS-PAGE gels (NuPAGE, Invitrogen). The samples were thenelectrotransferred to nitrocellulose membranes and immunoblotted withantibodies against p21 (Cell Signaling 1:1000), PDX1 (R&D systems1:500), NKX6.1 (DSHB 1:1000). Vinculin (Sigma-Aldrich 1:50000) was usedas a loading control. HRP conjugated secondary antibodies (JacksonImmunoResearch) were used for enhanced chemiluminescence detection (GEHealthcare).

Supplemental Data

Supplemental data, figures and tables can be found in Ameri et al. 2017and are incorporated herein in their entirety.

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1. A method of generating beta cells, comprising the steps of providinga starting cell population comprising at least one cell capable ofdifferentiation; wherein the cell capable of differentiation is apluripotent stem cell or a pancreatic progenitor cell expressing PDX1and NKX6.1, wherein: a. If the cell capable of differentiation is apluripotent stem cell, the method comprises the steps of: i) Incubatingsaid cell population in RPMI medium comprising Activin A and a glycogensynthase kinase (GSK3) inhibitor for a duration, thereby differentiatingat least part of the cell population into definitive endoderm cells; ii)Incubating the cell population of i) in RPMI medium comprisingB27−insulin, for a duration, thereby further differentiating the cellpopulation into definitive endoderm cells; iii) Incubating the cellpopulation of ii) in DMEM/F12 medium comprising B27+insulin and retinoicacid, for a duration, thereby differentiating at least part of the cellpopulation into gut tube cells; iv) incubating the cell population ofiii) in DMEM/F12 medium comprising B27+insulin and human FGF2, andoptionally human Noggin, for a duration, thereby differentiating atleast part of the cell population into posterior foregut cells; v)Incubating the cell population of iv) in DMEM/F12 medium comprisingB27+insulin,((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam)(TPB), and human Noggin for a duration, thereby differentiating at leastpart of the cell population into early pancreatic progenitor cells; andvi) Incubating the cell population of v) in DMEM/F12 medium comprisingB27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide, and human Nogginfor a duration, thereby differentiating at least part of the cellpopulation into mature pancreatic progenitor cells; and vii) Furtherincubating the cell population of vi) for an additional duration,thereby differentiating at least part of the cell population into betacells; or b. If the cell capable of differentiation is a pancreaticprogenitor cell, the method comprises the steps of: viii) Incubating thestarting cell population in DMEM/F12 medium comprising B27+insulin,Forskolin, Alk5 inhibitor, Nicotinamide, human Noggin and Rock inhibitorfor a duration; and ix) Incubating the cell population obtained in stepviii) in DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5inhibitor, Nicotinamide, human Noggin without Rock inhibitor for aduration.
 2. The method according to claim 1, wherein the incubation ofstep i) is for a duration of one day.
 3. The method according to any oneof the preceding claims, wherein the incubation of step ii) is for aduration of between 3 and 6 days.
 4. The method according to any one ofthe preceding claims, wherein the incubation of step iii) is for aduration of between 3 and 6 days.
 5. The method according to any one ofthe preceding claims, wherein the incubation of step iv) is for aduration of between 3 and 6 days.
 6. The method according to any one ofthe preceding claims, wherein the incubation of step v) is for aduration of between 3 and 6 days.
 7. The method according to any one ofthe preceding claims, wherein the incubation of step vi) is for aduration of between 3 and 6 days.
 8. The method according to any one ofthe preceding claims, wherein the incubation of step vii) is for aduration of between 7 and 23 days.
 9. The method according to any one ofthe preceding claims, wherein the incubation of step viii) is for aduration of between 1 and 2 days.
 10. The method according to any one ofthe preceding claims, wherein the incubation of step ix) is for aduration of between 7 and 14 days.
 11. The method according to any oneof the preceding claims, wherein the RPMI medium of step i) comprisesbetween 1 and 500 ng/mL Activin A, such as between 10 and 400 ng/mLActivin A, such as between 25 and 300 ng/mL Activin A, such as between50 and 200 ng/mL Activin A, such as between 75 and 150 ng/mL Activin A.12. The method according to claim 2, wherein the RPMI medium of step i)comprises 100 ng/mL Activin A.
 13. The method according to any one ofthe preceding claims, wherein the RPMI medium of step i) comprisesbetween 1 and 100 μM of a glycogen synthase kinase (GSK3) inhibitor suchas CHIR, preferably CHIR99021, such as between 1 and 75 μM of a GSK3inhibitor, such as between 1 and 50 μM of a GSK3 inhibitor, such asbetween 1 and 25 μM of a GSK3 inhibitor, such as between 1 and 10 μM ofa GSK3 inhibitor, such as between 1 and 5 μM of a GSK3 inhibitor, suchas 3 μM GSK3 inhibitor, for example 3 μM CHIR, preferably 3 μMCHIR99021.
 14. The method according to any one of the preceding claims,wherein the RPMI medium of step ii) comprises 1× B27−insulin.
 15. Themethod according to any one of the preceding claims, wherein theincubation of step ii) is for a duration of 3 days, 4 days, 5 days or 6days, preferably 4 days.
 16. The method according to any one of thepreceding claims, wherein the DMEM/F12 medium of step iii) comprises 1×B27+insulin.
 17. The method according to anyone of the preceding claims,wherein the DMEM/F12 medium of step iii) comprises between 0.5 and 10 μMretinoic acid, such as between 0.75 and 7.5 μM retinoic acid, such asbetween 1.0 and 5 μM retinoic acid, such as between 1.5 and 2.5 μMretinoic acid, such as 2 μM retinoic acid.
 18. The method according toany one of the preceding claims, wherein the incubation of step iii) isfor a duration of 3 days, 4 days, 5 days or 6 days, preferably 3 days.19. The method according to any one of the preceding claims, wherein thecell population obtained in step iii) is washed prior to step iv). 20.The method according to any one of the preceding claims, wherein theDMEM/F12 medium of step iv) comprises 1× B27+insulin.
 21. The methodaccording to any one of the preceding claims, wherein the DMEM/F12medium of step iv) comprises between 10 and 200 ng/mL human FGF2, suchas between 20 and 175 ng/mL human FGF2, such as between 30 and 150 ng/mLhuman FGF2, such as between 40 and 125 ng/mL human FGF2, such as between50 and 100 ng/mL human FGF2, such as between 55 and 90 ng/mL human FGF2,such as between 60 and 80 ng/mL human FGF2, such as between 60 and 70ng/mL human FGF2, such as 64 ng/mL human FGF2.
 22. The method accordingto any one of the preceding claims, wherein the DMEM/F12 medium of stepiv) comprises between 10 and 500 ng/mL human noggin, such as between 15and 400 ng/mL human noggin, such as between 20 and 300 ng/mL humannoggin, such as between 30 and 200 ng/mL human noggin, such as between40 and 100 ng/mL human noggin, such as between 45 and 75 ng/mL humannoggin, such as 50 ng/mL human noggin.
 23. The method according to anyone of the preceding claims, wherein the incubation of step iv) is for aduration of 3 days, 4 days, 5 days or 6 days, preferably 3 days.
 24. Themethod according to any one of the preceding claims, wherein theDMEM/F12 medium of step v) comprises 1× B27+insulin.
 25. The methodaccording to any one of the preceding claims, wherein the DMEM/F12medium of step v) comprises between 0.1 and 10 μM TPB, such as between0.2 and 7.5 μM TPB, such as between 0.3 and 5 μM TPB, such as between0.4 and 2.5 μM TPB, such as between 0.4 and 1 μM TPB, such as 0.5 μMTPB.
 26. The method according to any one of the preceding claims,wherein the DMEM/F12 medium of step v) comprises between 10 and 500ng/mL human noggin, such as between 25 and 400 ng/mL human noggin, suchas between 50 and 300 ng/mL human noggin, such as between 75 and 200ng/mL human noggin, such as between 75 and 150 ng/mL human noggin, suchas 100 ng/mL human noggin.
 27. The method according to any one of thepreceding claims, wherein the incubation of step v) is for a duration of3 days, 4 days, 5 days or 6 days, preferably 3 days.
 28. The methodaccording to any one of the preceding claims, wherein the DMEM/F12medium of steps vi), vii), viii) and/or ix) comprises 1× B27+insulin.29. The method according to any one of the preceding claims, wherein theDMEM/F12 medium of steps vi), vii), viii) or ix) comprises between 1 and500 μM Forskolin, such as between 2 and 400 μM Forskolin, such asbetween 3 and 300 μM Forskolin, such as between 4 and 200 μM Forskolin,such as between 5 and 100 μM Forskolin, such as between 6 and 75 μMForskolin, such as between 7 and 50 μM Forskolin, such as between 8 and25 μM Forskolin, such as between 9 and 15 μM Forskolin, such as 10 μMForskolin.
 30. The method according to any one of the preceding claims,wherein the DMEM/F12 medium of steps vi), vii), viii) or ix) comprisesbetween 1 and 100 μM Alk5 inhibitor, such as between 1.5 and 75 μM Alk5inhibitor, such as between 2 and 50 μM Alk5 inhibitor, such as between 3and 40 μM Alk5 inhibitor, such as between 3.5 and 30 μM Alk5 inhibitor,such as between 4.0 and 20 μM Alk5 inhibitor, such as between 4.5 and 10μM Alk5 inhibitor, such as 4.5 μM Alk5 inhibitor.
 31. The methodaccording to any one of the preceding claims, wherein the DMEM/F12medium of step vi), vii), viii) or ix) comprises between 1 and 100 mMNicotinamide, such as between 2 and 90 mM Nicotinamide, such as between3 and 80 mM Nicotinamide, such as between 4 and 70 mM Nicotinamide, suchas between 5 and 60 mM Nicotinamide, such as between 6 and 50 mMNicotinamide, such as between 7 and 40 mM Nicotinamide, such as between8 and 30 mM Nicotinamide, such as between 9 and 20 mM Nicotinamide, suchas 10 mM Nicotinamide.
 32. The method according to any one of thepreceding claims, wherein the DMEM/F12 medium of steps vi), vii), viii)or ix) comprises between 10 and 500 ng/mL human noggin, such as between25 and 400 ng/mL human noggin, such as between 50 and 300 ng/mL humannoggin, such as between 75 and 200 ng/mL human noggin, such as between75 and 150 ng/mL human noggin, such as 100 ng/mL human noggin.
 33. Themethod according to any one of the preceding claims, wherein theDMEM/F12 medium of step ix) comprises between 1 and 100 μM Rockinhibitor, such as between 2.5 and 75 μM Rock inhibitor, such as between5 and 50 μM Rock inhibitor, such as between 7.5 and 25 μM Rockinhibitor, such as between 10 and 15 μM Rock inhibitor.
 34. The methodaccording to any one of the preceding claims, wherein the incubation ofstep vi) is for a duration of 3 days, 4 days, 5 days or 6 days,preferably 3 days.
 35. The method according to any one of the precedingclaims, wherein the incubation of step vii) is for a duration of 7 days,8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days or 23 days.36. The method according to any one of the preceding claims, wherein theincubation of step ix) is for a duration of 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days or 14 days.
 37. The method according toany one of the preceding claims, wherein the medium is replaced by freshmedium on a daily basis in steps i) to v).
 38. The method according toany one of the preceding claims, wherein the medium is replaced by freshmedium every second day in steps vi), vii), viii) and ix).
 39. Themethod according to any one of the preceding claims, wherein the mediumof step i) does not comprise bovine serum albumin (BSA).
 40. The methodaccording to any one of the preceding claims, wherein the medium of stepii) does not comprise bovine serum albumin (BSA).
 41. The methodaccording to any one of the preceding claims, wherein the medium of stepiii) does not comprise bovine serum albumin (BSA).
 42. The methodaccording to any one of the preceding claims, wherein the medium of stepiv) does not comprise bovine serum albumin (BSA).
 43. The methodaccording to any one of the preceding claims, wherein the medium of stepv) does not comprise bovine serum albumin (BSA).
 44. The methodaccording to any one of the preceding claims, wherein the medium of stepvi) does not comprise bovine serum albumin (BSA).
 45. The methodaccording to any one of the preceding claims, wherein the medium of stepvii) does not comprise bovine serum albumin (BSA).
 46. The methodaccording to any one of the preceding claims, wherein the starting cellpopulation is a population of pluripotent stem cells.
 47. The methodaccording to claim 46, wherein the population of pluripotent stem cellsis a population of induced pluripotent stem cells, such as human inducedpluripotent stem cells.
 48. The method according to claim 46, whereinthe population of pluripotent stem cells is a population of embryonicstem cells, such as human embryonic stem cells.
 49. The method accordingto any one of the preceding claims, wherein the starting cell populationis a population of naïve stem cells, such as human naïve stem cells. 50.The method according to any one of the preceding claims, wherein thestarting cell population is a population of somatic cells.
 51. Themethod according to any one of the preceding claims, wherein thestarting cell population is a population of pancreatic progenitor cells.52. The method according to claim 51, wherein the method furthercomprises a step of enriching the starting cell population for cellsexpressing PDX1 and NKX6.1 between steps v) and vi) and/or between stepsvi) and vii).
 53. The method according to claim 52, wherein the step ofenriching the cell population for cells expressing PDX1 and NKX6.1comprises exposing the cell population to a first ligand which binds toa first marker specific for PDX1− cells and selecting the cells that donot bind to said first ligand from said cell population.
 54. The methodaccording to any one of claims 52 to 53, wherein the step of enrichingthe cell population for cells expressing PDX1 and NKX6.1 comprisesexposing the cell population to a second ligand which binds to a secondmarker specific for PDX1+ cells and selecting the cells that bind tosaid second ligand from the cells that do not bind to said secondligand, thereby enriching the cell population for PDX1+ cells.
 55. Themethod according to any one of claims 52 to 54, wherein the step ofenriching the cell population for cells expressing PDX1 and NKX6.1comprises exposing the cell population to a third ligand which binds toa third marker specific for PDX1+ NKX6.1+ cells and selecting the cellsthat bind to said third ligand from the cells that do not bind to saidthird ligand, thereby enriching the cell population for PDX1+ NKX6.1+cells.
 56. The method according to any one of claims 52 to 55, whereinthe step of enriching the cell population for cells expressing PDX1 andNKX6.1 comprises the steps of, in any order: a) exposing the cellpopulation to a first ligand which binds to a first marker specific forPDX1− cells and selecting the cells that do not bind to said firstligand from said cell population; and b) exposing the cell population toa second ligand which binds to a second marker specific for PDX1+ cellsand selecting the cells that bind to said second ligand from the cellsthat do not bind to said second ligand, thereby enriching the cellpopulation for PDX1+ cells.
 57. The method according to any one ofclaims 52 to 56, wherein the step of enriching the cell population forcells expressing PDX1 and NKX6.1 comprises the steps of, in any order:a) exposing the cell population to a first ligand which binds to a firstmarker specific for PDX1− cells and selecting the cells that do not bindto said first ligand from said cell population; and b) exposing the cellpopulation to a third ligand which binds to a third marker specific forPDX1+ NKX6.1+ cells and selecting the cells that bind to said thirdligand from the cells that do not bind to said third ligand, therebyenriching the cell population for PDX1+NKX6.1+ cells.
 58. The methodaccording to any one of claims 52 to 57, wherein the step of enrichingthe cell population for cells expressing PDX1 and NKX6.1 comprises thesteps of, in any order: a) exposing the cell population to a secondligand which binds to a second marker specific for PDX1+ cells andselecting the cells that bind to said second ligand from the cells thatdo not bind to said second ligand, thereby enriching the cell populationfor PDX1+ cells; and b) exposing the cell population to a third ligandwhich binds to a third marker specific for PDX1+ NKX6.1+ cells andselecting the cells that bind to said third ligand from the cells thatdo not bind to said third ligand, thereby enriching the cell populationfor PDX1+NKX6.1+ cells.
 59. The method according to any one of claims 52to 58, wherein the step of enriching the cell population for cellsexpressing PDX1 and NKX6.1 comprises the steps of, in any order: a)exposing the cell population to a first ligand which binds to a firstmarker specific for PDX1− cells and selecting the cells that do not bindto said first ligand from said cell population; and b) exposing the cellpopulation to a second ligand which binds to a second marker specificfor PDX1+ cells and selecting the cells that bind to said second ligandfrom the cells that do not bind to said second ligand, thereby enrichingthe cell population for PDX1+ cells; and c) exposing the cell populationto a third ligand which binds to a third marker specific for PDX1+NKX6.1+ cells and selecting the cells that bind to said third ligandfrom the cells that do not bind to said third ligand, thereby enrichingthe cell population for PDX1+NKX6.1+ cells.
 60. The method according toany one of claims 52 to 59, wherein at least one of the first, secondand third ligands is an antibody or fragment thereof.
 61. The methodaccording to any one of claims 52 to 60, wherein the antibody is amonoclonal or polyclonal antibody.
 62. The method according to any oneof claims 52 to 61, wherein at least one of the first, second and thirdligands binds to a cell surface marker of the pancreatic progenitorcell.
 63. The method according to any one of claims 52 to 62, wherein atleast one of the first, second and third ligands is conjugated to alabel.
 64. The method according to any one of claims 52 to 63, whereinthe expression of at least one of the first, second and third marker isdetected by flow cytometry.
 65. The method according to any one ofclaims 52 to 64, wherein the cells are removed or selected by flowcytometry.
 66. The method according to any one of claims 52 to 65,wherein the first ligand is an antibody or fragment thereof directedagainst CD49d.
 67. The method according to any one of claims 52 to 66,wherein the second ligand is an antibody or fragment thereof directedagainst a target selected from the group consisting of: FOLR1,CDH1/ECAD, F3/CD142, PDX1, FOXA2, EPCAM, HES1 and GATA4.
 68. The methodaccording to any one of claims 52 to 67, wherein the second ligand is anantibody or fragment thereof directed against FOLR1.
 69. The methodaccording to any one of claims 52 to 68, wherein the third ligand is anantibody or fragment thereof directed against a target selected from thegroup consisting of: GP2, SCN9A, MPZ, NAALADL2, KCNIP1, CALB1, SOX9,NKX6.2 and NKX6.1.
 70. The method according to any one of claims 52 to69, wherein the third ligand is an antibody or fragment thereof directedagainst a target selected from the group consisting of: GP2, SCN9A, MPZ,NAALADL2, KCNIP1 and CALB1.
 71. The method according to any one ofclaims 52 to 70, wherein the third ligand is an antibody or fragmentthereof directed against GP2.
 72. The method according to any one ofclaims 52 to 71, wherein the first ligand is an antibody or fragmentthereof directed against CD49d and the third ligand is an antibodydirected against GP2.
 73. The method according to any one of claims 52to 72, wherein the first ligand is an antibody or fragment thereofdirected against CD49d, the second ligand is an antibody or fragmentthereof directed against FOLR1 and the third ligand is an antibody orfragment thereof directed against GP2.
 74. The method according to anyone of the preceding claims, wherein if the starting population is apluripotent stem cell population, the method further comprises the stepof enriching the cell population obtained in step v) and/or in step vi)for cells expressing PDX1 and NKX6.1 as defined in any one of claims 53to
 74. 75. The method according to any one of the preceding claims,wherein the starting cell population is derived from cells isolated froman individual.
 76. The method according to any one of the precedingclaims, wherein at least one cell of the cell population obtained in anyof steps i) to iix) has the capability to differentiate further.
 77. Themethod according to any one of the preceding claims, wherein at leastone cell of the cell population obtained in any of steps i) to ix) hasthe capability to differentiate further into pancreatichormone-producing cells.
 78. The method according to any one of thepreceding claims, wherein the cell population obtained in step vii) orix) is capable of producing insulin.
 79. The method according to any oneof the preceding claims, wherein the cell population obtained in stepvii) or ix) is glucose-responsive.
 80. The method according to any oneof the preceding claims, wherein the cell population obtained in stepvii) or ix) can produce insulin-producing islet cells.
 81. A populationof cells obtainable by the method according to any one of the precedingclaims, for treatment of a metabolic disorder in an individual in needthereof.
 82. The population according to claim 81, wherein the cells arebeta cells.
 83. A method of treatment of a metabolic disorder in anindividual in need thereof, wherein the method comprises a step ofproviding a population of beta cells obtainable by the method accordingto any one of claims 1 to 80 and transplanting said population of betacells into said individual.
 84. The method according to claim 83,wherein the metabolic disorder is diabetes mellitus, such asinsulin-dependent diabetes mellitus, non-insulin-dependent diabetesmellitus, malnutrition-related diabetes mellitus, type 1 diabetes, type2 diabetes or unspecified diabetes mellitus.